Heterologous expression of Neisserial proteins

ABSTRACT

Alternative and improved approaches to the heterologous expression of the proteins of  Neisseria meningitidis  and  Neisseria gonorrhoeae  are disclosed. These approaches typically affect the level of expression, the ease of purification, the cellular localization, and/or the immunological properties of the expressed protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No. 14/244,806, filed Apr. 3, 2014; which is a Continuation of U.S. application Ser. No. 13/340,549, filed Dec. 29, 2011, now U.S. Pat. No. 8,703,914; which is a Divisional of U.S. application Ser. No. 12/825,210, filed Jun. 28, 2010, now U.S. Pat. No. 8,114,960; which is a Divisional of U.S. application Ser. No. 10/220,481, which claims an international filing date of Feb. 28, 2001, now U.S. Pat. No. 7,803,387; which is the National Phase of PCT Application No. PCT/IB2001/000452, filed Feb. 28, 2001; which claims the benefit of GB Application No. 0027675.8, filed Nov. 13, 2000; and GB Application No. 0004695.3, filed Feb. 28, 2000; all of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention is in the field of protein expression. In particular, it relates to the heterologous expression of proteins from Neisseria (e.g. N. gonorrhoeae or, preferably, N. meningitidis).

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 223002099802SubSeqList.txt, date recorded: Oct. 9, 2014,size: 505 KB).

BACKGROUND

International patent applications WO99/24578, WO99/36544, WO99/57280 and WO00/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhoeae. These proteins are typically described as being expressed in E. coli (i.e. heterologous expression) as either N-terminal GST-fusions or C-terminal His-tag fusions, although other expression systems, including expression in native Neisseria, are also disclosed.

It is an object of the present invention to provide alternative and improved approaches for the heterologous expression of these proteins. These approaches will typically affect the level of expression, the ease of purification, the cellular localisation of expression, and/or the immunological properties of the expressed protein.

DISCLOSURE

Nomenclature Herein

The 2166 protein sequences disclosed in WO99/24578, WO99/36544 and WO99/57280 are referred to herein by the following SEQ#numbers:

Application Protein sequences SEQ# herein WO99/24578 Even SEQ IDs 2-892 SEQ#s 1-446 WO99/36544 Even SEQ IDs 2-90 SEQ#s 447-491 WO99/57280 Even SEQ IDs 2-3020 SEQ#s 492-2001 Even SEQ IDs 3040-3114 SEQ#s 2002-2039 SEQ IDs 3115-3241 SEQ#s 2040-2166

In addition to this SEQ#numbering, the naming conventions used in WO99/24578, WO99/36544 and WO99/57280 are also used (e.g. ‘ORF4’, ‘ORF40’, ‘ORF40-1’ etc. as used in WO99/24578 and WO99/36544; ‘m919’, ‘g919’ and ‘a919’ etc. as used in WO99/57280).

The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [Science (2000) 287:1809-1815] are referred to herein as SEQ#s 2167-4326 [see also WO0/66791].

The term ‘protein of the invention’ as used herein refers to a protein comprising:

-   -   (a) one of sequences SEQ#s 1-4326; or     -   (b) a sequence having sequence identity to one of SEQ#s 1-4326;         or     -   (c) a fragment of one of SEQ#s 1-4326.

The degree of ‘sequence identity’ referred to in (b) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants [e.g. see WO00/66741]. Identity is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.

The ‘fragment’ referred to in (c) should comprise at least n consecutive amino acids from one of SEQ#s 1-4326 and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragment comprises an epitope from one of SEQ#s 1-4326. Preferred fragments are those disclosed in WO00/71574 and WO01/04316.

Preferred proteins of the invention are found in N. meningitidis serogroup B.

Preferred proteins for use according to the invention are those of serogroup B N. meningitidis strain 2996 or strain 394/98 (a New Zealand strain). Unless otherwise stated, proteins mentioned herein are from N. meningitidis strain 2996. It will be appreciated, however, that the invention is not in general limited by strain. References to a particular protein (e.g. ‘287’, ‘919’ etc.) may be taken to include that protein from any strain.

Non-Fusion Expression

In a first approach to heterologous expression, no fusion partner is used, and the native leader peptide (if present) is used. This will typically prevent any ‘interference’ from fusion partners and may alter cellular localisation and/or post-translational modification and/or folding in the heterologous host.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) no fusion partner is used, and (b) the protein's native leader peptide (if present) is used.

The method will typically involve the step of preparing an vector for expressing a protein of the invention, such that the first expressed amino acid is the first amino acid (methionine) of said protein, and last expressed amino acid is the last amino acid of said protein (i.e. the codon preceding the native STOP codon).

This approach is preferably used for the expression of the following proteins using the native leader peptide: 111, 149, 206, 225-1, 235, 247-1, 274, 283, 286, 292, 401, 406, 502-1, 503, 519-1, 525-1, 552, 556, 557, 570, 576-1, 580, 583, 664, 759, 907, 913, 920-1, 936-1, 953, 961, 983, 989, Orf4, Orf7-1, Orf9-1, Orf23, Or25, Orf37, Orf38, Orf40, Orf40.1, Orf40.2, Orf72-1, Orf76-1, Orf85-2, Orf91, Orf97-1, Orf119, Orf143.1, NMB0109 and NMB2050.The suffix ‘L’ used herein in the name of a protein indicates expression in this manner using the native leader peptide.

Proteins which are preferably expressed using this approach using no fusion partner and which have no native leader peptide include: 008, 105, 117-1, 121-1, 122-1, 128-1, 148, 216, 243, 308, 593, 652, 726, 926, 982, Orf83-1 and Orf143-1.

Advantageously, it is used for the expression of ORF25 or ORF40, resulting in a protein which induces better anti-bactericidal antibodies than GST- or His-fusions.

This approach is particularly suited for expressing lipoproteins.

Leader-Peptide Substitution

In a second approach to heterologous expression, the native leader peptide of a protein of the invention is replaced by that of a different protein. In addition, it is preferred that no fusion partner is used. Whilst using a protein's own leader peptide in heterologous hosts can often localise the protein to its ‘natural’ cellular location, in some cases the leader sequence is not efficiently recognised by the heterologous host. In such cases, a leader peptide known to drive protein targeting efficiently can be used instead.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's leader peptide is replaced by the leader peptide from a different protein and, optionally, (b) no fusion partner is used.

The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove nucleotides that encode the protein's leader peptide and to introduce nucleotides that encode a different protein's leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The expressed protein will consist of the replacement leader peptide at the N-terminus, followed by the protein of the invention minus its leader peptide.

The leader peptide is preferably from another protein of the invention (e.g. one of SEQ#s 1-4326), but may also be from an E. coli protein (e.g. the OmpA leader peptide) or an Erwinia carotovora protein (e.g. the PelB leader peptide), for instance.

A particularly useful replacement leader peptide is that of ORF4. This leader is able to direct lipidation in E. coli, improving cellular localisation, and is particularly useful for the expression of proteins 287, 919 and ΔG287. The leader peptide and N-terminal domains of 961 are also particularly useful.

Another useful replacement leader peptide is that of E. coli OmpA. This leader is able to direct membrane localisation of E. coli. It is particularly advantageous for the expression of ORF1, resulting in a protein which induces better anti-bactericidal antibodies than both fusions and protein expressed from its own leader peptide.

Another useful replacement leader peptide is MKKYLFSAA. (SEQ ID NO:621) This can direct secretion into culture medium, and is extremely short and active. The use of this leader peptide is not restricted to the expression of Neisserial proteins—it may be used to direct the expression of any protein (particularly bacterial proteins).

Leader-Peptide Deletion

In a third approach to heterologous expression, the native leader peptide of a protein of the invention is deleted. In addition, it is preferred that no fusion partner is used.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's leader peptide is deleted and, optionally, (b) no fusion partner is used.

The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove nucleotides that encode the protein's leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The first amino acid of the expressed protein will be that of the mature native protein.

This method can increase the levels of expression. For protein 919, for example, expression levels in E. coli are much higher when the leader peptide is deleted. Increased expression may be due to altered localisation in the absence of the leader peptide.

The method is preferably used for the expression of 919, ORF46, 961, 050-1, 760 and 287.

Domain-Based Expression

In a fourth approach to heterologous expression, the protein is expressed as domains. This may be used in association with fusion systems (e.g. GST or His-tag fusions).

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) at least one domain in the protein is deleted and, optionally, (b) no fusion partner is used.

The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove at least one domain from within the protein. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. Where no fusion partners are used, the first amino acid of the expressed protein will be that of a domain of the protein.

A protein is typically divided into notional domains by aligning it with known sequences in databases and then determining regions of the protein which show different alignment patterns from each other.

The method is preferably used for the expression of protein 287. This protein can be notionally split into three domains, referred to as A B & C (see FIG. 5). Domain B aligns strongly with IgA proteases, domain C aligns strongly with transferrin-binding proteins, and domain A shows no strong alignment with database sequences. An alignment of polymorphic forms of 287 is disclosed in WO00/66741.

Once a protein has been divided into domains, these can be (a) expressed singly (b) deleted from with the protein e.g. protein ABCD→ABD, ACD, BCD etc. or (c) rearranged e.g. protein ABC→ACB, CAB etc. These three strategies can be combined with fusion partners is desired.

ORF46 has also been notionally split into two domains—a first domain (amino acids 1-433) which is well-conserved between species and serogroups, and a second domain (amino acids 433-608) which is not well-conserved. The second domain is preferably deleted. An alignment of polymorphic forms of ORF46 is disclosed in WO00/66741.

Protein 564 has also been split into domains (FIG. 8), as have protein 961 (FIG. 12) and protein 502 (amino acids 28-167 of the MC58 protein).

Hybrid Proteins

In a fifth approach to heterologous expression, two or more (e.g. 3, 4, 5, 6 or more) proteins of the invention are expressed as a single hybrid protein. It is preferred that no non-Neisserial fusion partner (e.g. GST or poly-His) is used.

This offers two advantages. Firstly, a protein that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem. Secondly, commercial manufacture is simplified—only one expression and purification need be employed in order to produce two separately-useful proteins.

Thus the invention provides a method for the simultaneous heterologous expression of two or more proteins of the invention, in which said two or more proteins of the invention are fused (i.e. they are translated as a single polypeptide chain).

The method will typically involve the steps of: obtaining a first nucleic acid encoding a first protein of the invention; obtaining a second nucleic acid encoding a second protein of the invention; ligating the first and second nucleic acids. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.

Preferably, the constituent proteins in a hybrid protein according to the invention will be from the same strain.

The fused proteins in the hybrid may be joined directly, or may be joined via a linker peptide e.g. via a poly-glycine linker (i.e. G_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more) or via a short peptide sequence which facilitates cloning. It is evidently preferred not to join a ΔG protein to the C-terminus of a poly-glycine linker.

The fused proteins may lack native leader peptides or may include the leader peptide sequence of the N-terminal fusion partner.

The method is well suited to the expression of proteins orf1, orf4, orf25, orf40, Orf46/46.1, orf83, 233, 287, 292L, 564, 687, 741, 907, 919, 953, 961 and 983.

The 42 hybrids indicated by ‘X’ in the following table of form NH₂-A-B—COOH are preferred:

B A O ^(RF) 46.1 287 741 919 953 961 983 ORF46.1 X X X X X X 287 X X X X X X 741 X X X X X X 919 X X X X X X 953 X X X X X X 961 X X X X X X 983 X X X X X X

Preferred proteins to be expressed as hybrids are thus ORF46.1, 287, 741, 919, 953, 961 and 983. These may be used in their essentially full-length form, or poly-glycine deletions (AG) forms may be used (e.g. ΔG-287, AGTbp2, ΔG741, ΔG983 etc.), or truncated forms may be used (e.g. Δ1-287, Δ2-287 etc.), or domain-deleted versions may be used (e.g. 287B, 287C, 287BC, ORF46₁₋₄₃₃, ORF46₄₃₃₋₆₀₈, ORF46, 961c etc.).

Particularly preferred are: (a) a hybrid protein comprising 919 and 287; (b) a hybrid protein comprising 953 and 287; (c) a hybrid protein comprising 287 and ORF46.1; (d) a hybrid protein comprising ORF1 and ORF46.1; (e) a hybrid protein comprising 919 and ORF46.1; (f) a hybrid protein comprising ORF46.1 and 919; (g) a hybrid protein comprising ORF46.1, 287 and 919; (h) a hybrid protein comprising 919 and 519; and (i) a hybrid protein comprising ORF97 and 225. Further embodiments are shown in FIG. 14.

Where 287 is used, it is preferably at the C-terminal end of a hybrid; if it is to be used at the N-terminus, if is preferred to use a ΔG form of 287 is used (e.g. as the N-terminus of a hybrid with ORF46.1, 919, 953 or 961).

Where 287 is used, this is preferably from strain 2996 or from strain 394/98.

Where 961 is used, this is preferably at the N-terminus. Domain forms of 961 may be used.

Alignments of polymorphic forms of ORF46, 287, 919 and 953 are disclosed in WO00/66741. Any of these polymorphs can be used according to the present invention.

Temperature

In a sixth approach to heterologous expression, proteins of the invention are expressed at a low temperature.

Expressed Neisserial proteins (e.g. 919) may be toxic to E. coli, which can be avoided by expressing the toxic protein at a temperature at which its toxic activity is not manifested.

Thus the present invention provides a method for the heterologous expression of a protein of the invention, in which expression of a protein of the invention is carried out at a temperature at which a toxic activity of the protein is not manifested.

A preferred temperature is around 30° C. This is particularly suited to the expression of 919.

Mutations

As discussed above, expressed Neisserial proteins may be toxic to E. coli. This toxicity can be avoided by mutating the protein to reduce or eliminate the toxic activity. In particular, mutations to reduce or eliminate toxic enzymatic activity can be used, preferably using site-directed mutagenesis.

In a seventh approach to heterologous expression, therefore, an expressed protein is mutated to reduce or eliminate toxic activity.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which protein is mutated to reduce or eliminate toxic activity.

The method is preferably used for the expression of protein 907, 919 or 922. A preferred mutation in 907 is at Glu-117 (e.g. Glu→Gly); preferred mutations in 919 are at Glu-255 (e.g. Glu→Gly) and/or Glu-323 (e.g. Glu→Gly); preferred mutations in 922 are at Glu-164 (e.g. Glu→Gly), Ser-213 (e.g. Ser→Gly) and/or Asn-348 (e.g. Asn→Gly).

Alternative Vectors

In a eighth approach to heterologous expression, an alternative vector used to express the protein. This may be to improve expression yields, for instance, or to utilise plasmids that are already approved for GMP use.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which an alternative vector is used. The alternative vector is preferably pSM214, with no fusion partners. Leader peptides may or may not be included.

This approach is particularly useful for protein 953. Expression and localisation of 953 with its native leader peptide expressed from pSM214 is much better than from the pET vector.

pSM214 may also be used with: ΔG287, Δ2-287, Δ3-287, Δ4-287, Orf46.1, 961L, 961, 961(MC58), 961c, 961c-L, 919, 953 and ΔG287-Orf46.1.

Another suitable vector is pET-24b (Novagen; uses kanamycin resistance), again using no fusion partners. pET-24b is preferred for use with: ΔG287K, Δ2-287K, Δ3-287K, Δ4-287K, Orf46.1-K, Orf46A-K, 961-K (MC58), 961a-K, 961b-K, 961c-K, 961c-L-K, 961d-K, ΔG287-919-K, ΔG287-Orf46.1-K and ΔG287-961-K.

Multimeric Form

In a ninth approach to heterologous expression, a protein is expressed or purified such that it adopts a particular multimeric form.

This approach is particularly suited to protein 953. Purification of one particular multimeric form of 953 (the monomeric form) gives a protein with greater bactericidal activity than other forms (the dimeric form).

Proteins 287 and 919 may be purified in dimeric form.

Protein 961 may be purified in a 180 kDa oligomeric form (e.g. a tetramer).

Lipidation

In a tenth approach to heterologous expression, a protein is expressed as a lipidated protein.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which the protein is expressed as a lipidated protein.

This is particularly useful for the expression of 919, 287, ORF4, 406, 576-1, and ORF25. Polymorphic forms of 919, 287 and ORF4 are disclosed in WO00/66741.

The method will typically involve the use of an appropriate leader peptide without using an N-terminal fusion partner.

C-Terminal Deletions

In an eleventh approach to heterologous expression, the C-terminus of a protein of the invention is mutated. In addition, it is preferred that no fusion partner is used.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's C-terminus region is mutated and, optionally, (b) no fusion partner is used.

The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to mutate nucleotides that encode the protein's C-terminus portion. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The first amino acid of the expressed protein will be that of the mature native protein.

The mutation may be a substitution, insertion or, preferably, a deletion.

This method can increase the levels of expression, particularly for proteins 730, ORF29 and ORF46. For protein 730, a C-terminus region of around 65 to around 214 amino acids may be deleted; for ORF46, the C-terminus region of around 175 amino acids may be deleted; for ORF29, the C-terminus may be deleted to leave around 230-370 N-terminal amino acids.

Leader Peptide Mutation

In a twelfth approach to heterologous expression, the leader peptide of the protein is mutated. This is particularly useful for the expression of protein 919.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which the protein's leader peptide is mutated.

The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; and manipulating said nucleic acid to mutate nucleotides within the leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.

Poly-Glycine Deletion

In a thirteenth approach to heterologous expression, poly-glycine stretches in wild-type sequences are mutated. This enhances protein expression.

The poly-glycine stretch has the sequence (Gly)_(n), where n≧4 (e.g. 5, 6, 7, 8, 9 or more). This stretch is mutated to disrupt or remove the (Gly)_(n). This may be by deletion (e.g. CGGGGS (SEQ ID NO:622)→CGGGS (SEQ ID NO:623), CGGS (SEQ ID NO:624), CGS or CS), by substitution (e.g. CGGGGS (SEQ ID NO:622)→CGXGGS (SEQ ID NO:625), CGXXGS (SEQ ID NO:626), CGXGXS (SEQ ID NO:627) etc.), and/or by insertion (e.g. CGGGGS (SEQ ID NO:622)→CGGXGGS (SEQ ID NO:628), CGXGGGS (SEQ ID NO:629), etc.).

This approach is not restricted to Neisserial proteins—it may be used for any protein (particularly bacterial proteins) to enhance heterologous expression. For Neisserial proteins, however, it is particularly suitable for expressing 287, 741, 983 and Tbp2. An alignment of polymorphic forms of 287 is disclosed in WO00/66741.

Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) a poly-glycine stretch within the protein is mutated.

The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; and manipulating said nucleic acid to mutate nucleotides that encode a poly-glycine stretch within the protein sequence. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.

Conversely, the opposite approach (i.e. introduction of poly-glycine stretches) can be used to suppress or diminish expression of a given heterologous protein.

Heterologous Host

Whilst expression of the proteins of the invention may take place in the native host (i.e. the organism in which the protein is expressed in nature), the present invention utilises a heterologous host. The heterologous host may be prokaryotic or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonenna typhimurium, Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea, Mycobateria (e.g. M. tuberculosis), yeast etc.

Vectors etc.

As well as the methods described above, the invention provides (a) nucleic acid and vectors useful in these methods (b) host cells containing said vectors (c) proteins expressed or expressable by the methods (d) compositions comprising these proteins, which may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions (e) these compositions for use as medicaments (e.g. as vaccines) or as diagnostic reagents (f) the use of these compositions in the manufacture of (1) a medicament for treating or preventing infection due to Neisserial bacteria (2) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria, and/or (3) a reagent which can raise antibodies against Neisserial bacteria and (g) a method of treating a patient, comprising administering to the patient a therapeutically effective amount of these compositions.

Sequences

The invention also provides a protein or a nucleic acid having any of the sequences set out in the following examples. It also provides proteins and nucleic acid having sequence identity to these. As described above, the degree of ‘sequence identity’ is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).

Furthermore, the invention provides nucleic acid which can hybridise to the nucleic acid disclosed in the examples, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).

The invention also provides nucleic acid encoding proteins according to the invention.

It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).

Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).

In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a construct used to express orf1 protein using a heterologous leader peptide.

FIG. 2 shows a construct used to express 287 protein using a heterologous leader peptide.

FIG. 3A-FIG. 3E show expression data for ORF1. FIG. 3A shows purification of ORF1.

FIG. 3B shows Western blot analysis. FIG. 3C shows the results of a bactericidal assay with ORF1. FIG. 3D shows FACS analysis. FIG. 3E shows the results of an ELISA assay.

FIG. 4A-FIG. 4E show expression data for protein 961. FIG. 4A shows purification of protein 961. FIG. 4B shows Western blot analysis. FIG. 4C shows the results of a bactericidal assay with protein 961. FIG. 4D shows FACS analysis. FIG. 4E shows the results of an ELISA assay.

FIG. 5 shows domains of protein 287 and FIGS. 6 & 7 (SEQ ID NO:619 and 620) show deletions within domain A.

FIG. 6 shows deletions within domain A of protein 287.

FIG. 7 shows specific deletions within domain A of protein 287.

FIG. 8 shows domains of protein 564.

FIG. 9 shows the PhoC reporter gene driven by the 919 leader peptide.

FIG. 10A-FIG. 10B show the results obtained using mutants of the 919 leader peptide driving the PhoC reporter. FIG. 10A shows results for control, phoC_(wt), 9phoC, 9L1a, 9l1d, 9L1f, and 9S1e. FIG. 10B shows results for control, phoC_(wt), 9phoC, 9S1b, 9S1c, and 9S1i.

FIG. 11A-FIG. 11B show insertion mutants of protein 730. FIG. 11A shows 730-C1.

FIG. 11B shows 730-C2.

FIG. 12 shows domains of protein 961.

FIG. 13 shows SDS-PAGE of ΔG proteins. Dots show the main recombinant product.

FIG. 14A-FIG. 14Z show 26 hybrid proteins according to the invention. FIG. 14A shows ΔG287-919. FIG. 14B shows ΔG287-953. FIG. 14C shows ΔG287-961. FIG. 14D shows ΔG287NZ-919. FIG. 14E shows ΔG287NZ-953. FIG. 14F shows ΔG287NZ-961. FIG. 14G shows ΔG983-ORF46.1. FIG. 14H shows ΔG983-741. FIG. 14I shows ΔG983-961.FIG. 14J shows ΔG983-961c. FIG. 14K shows ΔG741-961. FIG. 14L shows ΔG741-961c. FIG. 14M shows ΔG741-983. FIG. 14N shows ΔG741-ORF46.1. FIG. 14O shows ORF46.1-741. FIG. 14P shows ORF46.1-961. FIG. 14Q shows ORF46.1-961c. FIG. 14R shows 961-ORF46.1. FIG. 14S shows 961-741. FIG. 14T shows 961-983. FIG. 14U shows 961c-ORF46.1. FIG. 14V shows 961c-741. FIG. 14W shows 961c-983. FIG. 14X shows 961cL-ORF46.1. FIG. 14Y shows 961cL-741. FIG. 14Z shows 961cL-983.

MODES FOR CARRYING OUT THE INVENTION Example 1 919 and its Leader Peptide

Protein 919 from N. meningitidis (serogroup B, strain 2996) has the following sequence (SEQ ID NO:1):

  1 MKKYLFRAAL YGIAAAILAA CQSKSIQTFP QPDTSVINGP DRPVGIPDPA  51 GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV 101 CAQAFQTPVH SFQAKQFFER YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR 151 RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT 201 HTADLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA 251 EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMADKGYL 301 KLGQTSMQGI KAYMRQNPQR LAEVLGQNPS YIFFRELAGS SNDGPVGALG 351 TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG 401 AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P*

The leader peptide is underlined.

The sequences of 919 from other strains can be found in FIGS. 7 and 18 of WO00/66741.

Example 2 of WO99/57280 discloses the expression of protein 919 as a His-fusion in E. coli. The protein is a good surface-exposed immunogen.

Three alternative expression strategies were used for 919:

-   -   1) 919 without its leader peptide (and without the mature         N-terminal cysteine) and without any fusion partner         (‘919^(untagged)’) (SEQ ID NO:2):

  1 QSKSIQTFP QPDTSVINGP DRPVGIPDPA GTTVGGGGAV YTVVPHLSLP  50 HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV CAQAFQTPVH SFQAKQFFER 100 YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR RTAQARFPIY GIPDDFISVP 150 LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT HTADLSRFPI TARTTAIKGR 200 FEGSRFLPYH TRNQINGGAL DGKAPILGYA EDPVELFFMH IQGSGRLKTP 250 SGKYIRIGYA DKNEHPYVSI GRYMADKGYL KLGQTSMQGI KAYMRQNPQR 300 LAEVLGQNPS YIFFRELAGS SNDGPVGALG TPLMGEYAGA VDRHYITLGA 350 PLFVATAHPV TRKALNRLIM AQDTGSAIKG AVRVDYFWGY GDEAGELAGK 400 QKTTGYVWQL LPNGMKPEYR P*

-   -   The leader peptide and cysteine were omitted by designing the         5′-end amplification primer downstream from the predicted leader         sequence.     -   2) 919 with its own leader peptide but without any fusion         partner (‘919L’); and     -   3) 919 with the leader peptide (MKTFFKTLSAAALALILAA (SEQ ID NO:         630)) from ORF4 (‘919LOrf4’) (SEQ ID NO:3).

  1 MKTFFKTLS AAALALILAA CQSKSIQTFP QPDTSVINGP DRPVGIPDPA  50 GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV 100 CAQAFQTPVH SFQAKQFFER YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR 150 RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT 200 HTADLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA 250 EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMADKGYL 300 KLGQTSMQGI KSYMRQNPQR LAEVLGQNPS YIFFRELAGS SNDGPVGALG 350 TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG 400 AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P*

-   -   To make this construct, the entire sequence encoding the ORF4         leader peptide was included in the 5′-primer as a tail (primer         919Lorf4 For). A NheI restriction site was generated by a double         nucleotide change in the sequence coding for the ORF4 leader (no         amino acid changes), to allow different genes to be fused to the         ORF4 leader peptide sequence. A stop codon was included in all         the 3′-end primer sequences.

All three forms of the protein were expressed and could be purified.

The ‘919L’ and ‘919LOrf4’ expression products were both lipidated, as shown by the incorporation of [3-H]-palmitate label. 919^(untagged) did not incorporate the ³H label and was located intracellularly.

919LOrf4 could be purified more easily than 919L. It was purified and used to immunise mice. The resulting sera gave excellent results in FACS and ELISA tests, and also in the bactericidal assay. The lipoprotein was shown to be localised in the outer membrane.

919^(untagged) gave excellent ELISA titres and high serum bactericidal activity. FACS confirmed its cell surface location.

Example 2 919 and Expression Temperature

Growth of E. coli expressing the 919LOrf4 protein at 37° C. resulted in lysis of the bacteria. In order to overcome this problem, the recombinant bacteria were grown at 30° C. Lysis was prevented without preventing expression.

Example 3 Mutation of 907, 919 and 922

It was hypothesised that proteins 907, 919 and 922 are murein hydrolases, and more particularly lytic transglycosylases. Murein hydrolases are located on the outer membrane and participate in the degradation of peptidoglycan.

The purified proteins 919^(untagged), 919Lorf4, 919-His (i.e. with a C-terminus His-tag) and 922-His were thus tested for murein hydrolase activity [Ursinus & Holtje (1994) J. Bact. 176:338-343]. Two different assays were used, one determining the degradation of insoluble murein sacculus into soluble muropeptides and the other measuring breakdown of poly(MurNAc-GlcNAc)_(n>30) glycan strands.

The first assay uses murein sacculi radiolabelled with meso-2,6-diamino-3,4,5-[³H]pimelic acid as substrate. Enzyme (3-10 μg total) was incubated for 45 minutes at 37° C. in a total volume of 100 μl comprising 10 mM Tris-maleate (pH 5.5), 10 mM MgCl₂, 0.2% v/v Triton X-100 and [³H]A₂pm labelled murein sacculi (about 10000 cpm). The assay mixture was placed on ice for 15 minutes with 1001 of 1% w/v N-acetyl-N,N,N-trimethylammonium for 15 minutes and precipitated material pelleted by centrifugation at 10000 g for 15 minutes. The radioactivity in the supernatant was measured by liquid scintillation counting. E. coli soluble lytic transglycosylase Slt70 was used as a positive control for the assay; the negative control comprised the above assay solution without enzyme.

All proteins except 919-His gave positive results in the first assay.

The second assay monitors the hydrolysis of poly(MurNAc-GlcNAc)glycan strands. Purified strands, poly(MurNAc-GlcNAc)_(n>30) labelled with N-acetyl-D-1-[³H]glucosamine were incubated with 3 μg of 919L in 10 mM Tris-maleate (pH 5.5), 10 mM MgCl₂ and 0.2% v/v Triton X-100 for 30 min at 37° C. The reaction was stopped by boiling for 5 minutes and the pH of the sample adjusted to about 3.5 by addition of 10 μl of 20% v/v phosphoric acid. Substrate and product were separated by reversed phase HPLC on a NUCLEOSIL® 300 C₁₈ column (an octadecyl modified silica phase for HPLC) as described by Harz et. al. [Anal. Biochem. (1990) 190:120-128]. The E. coli lytic transglycosylase Mlt A was used as a positive control in the assay. The negative control was performed in the absence of enzyme.

By this assay, the ability of 919LOrf4 to hydrolyse isolated glycan strands was demonstrated when anhydrodisaccharide subunits were separated from the oligosaccharide by HPLC.

Protein 919Lorf4 was chosen for kinetic analyses. The activity of 919Lorf4 was enhanced 3.7-fold by the addition of 0.2% v/v Triton X-100 in the assay buffer. The presence of Triton X-100 had no effect on the activity of 919^(untagged). The effect of pH on enzyme activity was determined in Tris-Maleate buffer over a range of 5.0 to 8.0. The optimal pH for the reaction was determined to be 5.5. Over the temperature range 18° C. to 42° C., maximum activity was observed at 37° C. The effect of various ions on murein hydrolase activity was determined by performing the reaction in the presence of a variety of ions at a final concentration of 10 mM. Maximum activity was found with Mg²⁺, which stimulated activity 2.1-fold, Mn²⁺ and Ca²⁺ also stimulated enzyme activity to a similar extent while the addition Ni²⁺ and EDTA had no significant effect. In contrast, both Fe²⁺ and Zn²⁺ significantly inhibited enzyme activity.

The structures of the reaction products resulting from the digestion of unlabelled E. coli murein sacculus were analysed by reversed-phase HPLC as described by Glauner [Anal. Biochem. (1988) 172:451-464]. Murein sacculi digested with the muramidase Cellosyl were used to calibrate and standardise the Hypersil ODS column. The major reaction products were 1,6 anhydrodisaccharide tetra and tri peptides, demonstrating the formation of 1,6 anhydromuraminic acid intramolecular bond.

These results demonstrate experimentally that 919 is a murein hydrolase and in particular a member of the lytic transglycosylase family of enzymes. Furthermore the ability of 922-His to hydrolyse murein sacculi suggests this protein is also a lytic transglycosylase.

This activity may help to explain the toxic effects of 919 when expressed in E. coli.

In order to eliminate the enzymatic activity, rational mutagenesis was used. 907, 919 and 922 show fairly low homology to three membrane-bound lipidated murein lytic transglycosylases from E. coli:

-   -   919 (441aa) is 27.3% identical over 440aa overlap to E. coli         MLTA (P46885);     -   922 (369aa) is 38.7% identical over 310aa overlap to E. coli         MLTB (P41052); and     -   907-2 (207aa) is 26.8% identical over 149aa overlap to E. coli         MLTC (P52066).

907-2 also shares homology with E. coli MLTD (P23931) and Slt70 (P03810), a soluble lytic transglycosylase that is located in the periplasmic space. No significant sequence homology can be detected among 919, 922 and 907-2, and the same is true among the corresponding MLTA, MLTB and MLTC proteins.

Crystal structures are available for Slt70 [1QTEA; 1QTEB; Thunnissen et al. (1995) Biochemistry 34:12729-12737] and for Slt35 [1LTM; 1QUS; 1QUT; van Asselt et al. (1999) Structure Fold Des 7:1167-80] which is a soluble form of the 40 kDa MLTB.

The catalytic residue (a glutamic acid) has been identified for both Slt70 and MLTB.

In the case of Slt70, mutagenesis studies have demonstrated that even a conservative substitution of the catalytic Glu505 with a glutamine (Gln) causes the complete loss of enzymatic activity. Although Slt35 has no obvious sequence similarity to Slt70, their catalytic domains shows a surprising similarity. The corresponding catalytic residue in MLTB is Glu162.

Another residue which is believed to play an important role in the correct folding of the enzymatic cleft is a well-conserved glycine (Gly) downstream of the glutamic acid. Recently, Terrak et al. [Mol. Microbiol. (1999) 34:350-64] have suggested the presence of another important residue which is an aromatic amino acid located around 70-75 residues downstream of the catalytic glutamic acid.

Sequence alignment of Slt70 (SEQ ID NO:5) with 907-2 (SEQ ID NO:4) and of MLTB (SEQ ID NO:7) with 922 (SEQ ID NO:6) were performed in order to identify the corresponding catalytic residues in the MenB antigens.

The two alignments in the region of the catalytic domain are reported below:

907-2/Slt70:

922/MLTB

From these alignments, it results that the corresponding catalytic glutamate in 907-2 is Glu117, whereas in 922 is Glu164. Both antigens also share downstream glycines that could have a structural role in the folding of the enzymatic cleft (in bold), and 922 has a conserved aromatic residue around 70aa downstream (in bold).

In the case of protein 919, no 3D structure is available for its E. coli homologue MLTA, and nothing is known about a possible catalytic residue. Nevertheless, three amino acids in 919 (SEQ ID NO:8) are predicted as catalytic residues by alignment with MLTA (SEQ ID NO:9):

919/MLTA

The three possible catalytic residues are shown by the symbol ▾:

-   1) Glu255 (Asp in MLTA), followed by three conserved glycines     (Gly263, Gly265 and Gly272) and three conserved aromatic residues     located approximately 75-77 residues downstream. These downstream     residues are shown by □. -   2) Glu323 (conserved in MLTA), followed by 2 conserved glycines     (Gly347 and Gly355) and two conserved aromatic residues located     84-85 residues downstream (Tyr406 or Phe407). These downstream     residues are shown by ⋄. -   3) Asp362 (instead of the expected Glu), followed by one glycine     (Gly 369) and a conserved aromatic residue (Trp428). These     downstream residues are shown by ◯.

Alignments of polymorphic forms of 919 are disclosed in WO00/66741.

Based on the prediction of catalytic residues, three mutants of the 919 and one mutant of 907, containing each a single amino acid substitution, have been generated. The glutamic acids in position 255 and 323 and the aspartic acids in position 362 of the 919 protein and the glutamic acid in position 117 of the 907 protein, were replaced with glycine residues using PCR-based SDM. To do this, internal primers containing a codon change from Glu or Asp to Gly were designed:

SEQ  Codon Primers ID NO Sequences change 919-E255 for 10 CGAAGACCCCGTCGgtCTTTTTTTTATG GAA → Ggt 919-E255 rev 11 GTGCATAAAAAAAAGacCGACGGGGTCT 919-E323 for 12 AACGCCTCGCCGgtGTTTTGGGTCA GAA → Ggt 919-E323 rev 13 TTTGACCCAAAACacCGGCGAGGCG 919-D362 for 14 TGCCGGCGCAGTCGgtCGGCACTACA GAC → Ggt 919-D362 rev 15 TAATGTAGTGCCGacCGACTGCGCCG 907-E117 for 16 TGATTGAGGTGGgtAGCGCGTTCCG GAA → Ggt 907-E117 rev 17 GGCGGAACGCGCTacCCACCTCAAT

-   -   Underlined nucleotides code for glycine; the mutated nucleotides         are in lower case.

To generate the 919-E255, 919-E323 and 919-E362 mutants, PCR was performed using 20 ng of the pET 919-LOrf4 DNA as template, and the following primer pairs:

-   -   1) Orf4L for/919-E255 rev     -   2) 919-E255 for/919L rev     -   3) Orf4L for/919-E323 rev     -   4) 919-E323 for/919L rev     -   5) Orf4L for/919-D362 rev     -   6) 919-D362 for/919L rev

The second round of PCR was performed using the product of PCR 1-2, 3-4 or 5-6 as template, and as forward and reverse primers the “Orf4L for” and “919L rev” respectively.

For the mutant 907-E117, PCR have been performed using 200 ng of chromosomal DNA of the 2996 strain as template and the following primer pairs:

-   -   7) 907L for/907-E117 rev     -   8) 907-E117 for/907L rev

The second round of PCR was performed using the products of PCR 7 and 8 as templates and the oligos “907L for” and “907L rev” as primers.

The PCR fragments containing each mutation were processed following the standard procedure, digested with NdeI and XhoI restriction enzymes and cloned into pET-21b+ vector. The presence of each mutation was confirmed by sequence analysis.

Mutation of Glu117 to Gly in 907 is carried out similarly, as is mutation of residues Glu164, Ser213 and Asn348 in 922.

The E255G mutant of 919 shows a 50% reduction in activity; the E323G mutant shows a 70% reduction in activity; the E362G mutant shows no reduction in activity.

Example 4 Multimeric Form

287-GST, 919^(untagged) and 953-His were subjected to gel filtration for analysis of quaternary structure or preparative purposes. The molecular weight of the native proteins was estimated using either FPLC Superose 12 (H/R 10/30) or SUPERDEX™75 gel filtration columns (prepacked columns, Pharmacia). The buffers used for chromatography for 287, 919 and 953 were 50 mM Tris-HCQ (pH 8.0), 20 mM Bicine (pH 8.5) and 50 mM Bicine (pH 8.0), respectively.

Additionally each buffer contained 150-200 mM NaCl and 10% v/v glycerol. Proteins. were dialysed against the appropriate buffer and applied in a volume of 200 μl. Gel filtration was performed with a flow rate of 0.5-2.0 ml/min and the eluate monitored at 280 nm. Fractions were collected and analysed by SDS-PAGE. Blue dextran 2000 and the molecular weight standards ribonuclease A, chymotrypsin A ovalbumin, albumin (Pharmacia) were used to calibrate the column. The molecular weight of the sample was estimated from a calibration curve of K_(av) vs. log M_(T) of the standards. Before gel filtration, 287-GST was digested with thrombin to cleave the GST moiety.

The estimated molecular weights for 287, 919 and 953-His were 73 kDa, 47 kDa and 43 kDa respectively. These results suggest 919 is monomeric while both 287 and 953 are principally dimeric in their nature. In the case of 953-His, two peaks were observed during gel filtration. The major peak (80%) represented a dimeric conformation of 953 while the minor peak (20%) had the expected size of a monomer. The monomeric form of 953 was found to have greater bactericidal activity than the dimer.

Example 5 pSM214 and pET24b Vectors

953 protein with its native leader peptide and no fusion partners was expressed from the pET vector and also from pSM214 [Velati Bellini et al. (1991) J. Biotechnol. 18, 177-192].

The 953 sequence was cloned as a full-length gene into pSM214 using the E. coli MM294-1 strain as a host. To do this, the entire DNA sequence of the 953 gene (from ATG to the STOP codon) was amplified by PCR using the following primers:

(SEQ ID NO: 18) 953L for/2 CCGGAATTCTTATGAAAAAAATCATCTTCGCCGC Eco RI (SEQ ID NO: 19) 953L rev/2 GCCCAAGCTTTTATTGTTTGGCTGCCTCGATT Hind III which contain EcoRI and HindIII restriction sites, respectively. The amplified fragment was digested with EcoRI and HindIII and ligated with the pSM214 vector digested with the same two enzymes. The ligated plasmid was transformed into E. coli MM294-1 cells (by incubation in ice for 65 minutes at 37° C.) and bacterial cells plated on LB agar containing 20 μg/ml of chloramphenicol.

Recombinant colonies were grown over-night at 37° C. in 4 ml of LB broth containing 20 μg/ml of chloramphenicol; bacterial cells were centrifuged and plasmid DNA extracted as and analysed by restriction with EcoRI and HindIII. To analyse the ability of the recombinant colonies to express the protein, they were inoculated in LB broth containing 20 μg/ml of chloramphenicol and let to grown for 16 hours at 37° C. Bacterial cells were centrifuged and resuspended in PBS. Expression of the protein was analysed by SDS-PAGE and Coomassie Blue staining.

Expression levels were unexpectedly high from the pSM214 plasmid.

Oligos used to clone sequences into pSM-214 vectors were as follows:

ΔG287 Fwd CCGGAATTCTTATG-TCGCCCGATGTTAAATCGGCGGA SEQ ID NO: 20 EcoRI (pSM-214) Rev GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG SEQ ID NO: 21 HindIII Δ2 287 Fwd CCGGAATTCTTATG-AGCCAAGATATGGCGGCAGT SEQ ID NO: 22 EcoRI (pSM-214) Rev GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG SEQ ID NO: 23 HindIII Δ3 287 Fwd CCGGAATTCTTATG-TCCGCCGAATCCGCAAATCA SEQ ID NO: 24 EcoRI (pSM-214) Rev GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG SEQ ID NO: 25 HindIII Δ4 287 Fwd CCGGAATTCTTATG-GGAAGGGTTGATTTGGCTAATG SEQ ID NO: 26 EcoRI (pSM-214) Rev GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG SEQ ID NO: 27 HindIII Orf46.1 Fwd CCGGAATTCTTATG-TCAGATTTGGCAAACGATTCTT SEQ ID NO: 28 EcoRI (pSM-214) Rev GCCCAAGCTT-TTACGTATCATATTTCACGTGCTTC SEQ ID NO: 29 HindIII ΔG 287- Fwd CCGGAATTCTTATG-TCGCCCGATGTTAAATCGGCGGA SEQ ID NO: 30 EcoRI Orf46.1 Rev GCCCAAGCTT-TTACGTATCATATTTCACGTGCTTC SEQ ID NO: 31 HindIII (pSM-214) 919 Fwd CCGGAATTCTTATG-CAAAGCAAGAGCATCCAAACCT SEQ ID NO: 32 EcoRI (pSM-214) Rev GCCCAAGCTT-TTACGGGCGGTATTCGGGCT SEQ ID NO: 33 HindIII 961L Fwd CCGGAATTCATATG-AAACACTTTCCATCC SEQ ID NO: 34 EcoRI (pSM-214) Rev GCCCAAGCTT-TTACCACTCGTAATTGAC SEQ ID NO: 35 HindIII 961 Fwd CCGGAATTCATATG-GCCACAAGCGACGAC SEQ ID NO: 36 EcoRI (pSM-214) Rev GCCCAAGCTT-TTACCACTCGTAATTGAC SEQ ID NO: 37 HindIII 961c L Fwd CCGGAATTCTTATG-AAACACTTTCCATCC SEQ ID NO: 38 EcoRI pSM-214 Rev GCCCAAGCTT-TCAACCCACGTTGTAAGGTTG SEQ ID NO: 39 HindIII 961c Fwd CCGGAATTCTTATG-GCCACAAACGACGACG SEQ ID NO: 40 EcoRI pSM-214 Rev GCCCAAGCTT-TCAACCCACGTTGTAAGGTTG SEQ ID NO: 41 HindIII 953 Fwd CCGCAATTCTTATG-GCCACCTACAAAGTGGACGA SEQ ID NO: 42 EcoRI (pSM-214) Rev GCCCAAGCTT-TTATTGTTTGGCTGCCTCGATT SEQ ID NO: 43 HindIII

These sequences were manipulated, cloned and expressed as described for 953L.

For the pET-24 vector, sequences were cloned and the proteins expressed in pET-24 as described below for pET21. pET2 has the same sequence as pET-21, but with the kanamycin resistance cassette instead of ampicillin cassette.

Oligonucleotides used to clone sequences into pET-24b vector were:

ΔG 287 K Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC ^(§) SEQ ID NO: 44 NheI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC * SEQ ID NO: 45 XhoI Δ2 287 K Fwd CGCGGATCCGCTAGC-CAAGATATGGCGGCAGT ^(§) SEQ ID NO: 46 NheI Δ3 287 K Fwd CGCGGATCCGCTAGC-GCCGAATCCGCAAATCA ^(§) SEQ ID NO: 47 NheI Δ4 287 K Fwd CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG ^(§) SEQ ID NO: 48 NheI Orf46.1 K Fwd GGGAATTCCATATG-GGCATTTCCCGCAAAATATC SEQ ID NO: 49 NdeI Rev CCCGCTCGAG-TTACGTATCATATTTCACGTGC SEQ ID NO: 50 XhoI Orf46A K Fwd GGGAATTCCATATG-GGCATTTCCCGCAAAATATC SEQ ID NO: 51 NdeI Rev CCCGCTCGAG-TTATTCTATGCCTTGTGCGGCAT SEQ ID NO: 52 XhoI 961 K Fwd CGCGGATCCCATATG-GCCACAAGCGACGACGA SEQ ID NO: 53 NdeI (MCS8) Rev CCCGCTCGAG-TTACCACTCGTAATTGAC SEQ ID NO: 54 XhoI 961a K Fwd CGCGGATCCCATATG-GCCACAAACGACG SEQ ID NO: 55 NdeI Rev CCCGCTCGAG-TCATTTAGCAATATTATCTTTGTTC SEQ ID NO: 56 XhoI 961b K Fwd CGCGGATCCCATATG-AAAGCAAACAGTGCCGAC SEQ ID NO: 57 NdeI Rev CCCGCTCGAG-TTACCACTCGTAATTGAC SEQ ID NO: 58 XhoI 961c K Fwd CGCGGATCCCATATG-GCCACAAACGACG SEQ ID NO: 59 NdeI Rev CCCGCTCGAG-TTAACCCACGTTGTAAGGT SEQ ID NO: 60 XhoI 961cL K Fwd CGCGGATCCCATATG-ATGAAACACTTTCCATCC SEQ ID NO: 61 NdeI Rev CCCGCTCGAG-TTAACCCACGTTGTAAGGT SEQ ID NO: 62 XhoI 961d K Fwd CGCGGATCCCATATG-GCCACAAACGACG SEQ ID NO: 63 NdeI Rev CCCGCTCGAG-TCAGTCTGACACTGTTTTATCC SEQ ID NO: 64 XhoI ΔG 287- Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC SEQ ID NO: 65 NheI 919 K Rev CCCGCTCGAG-TTACGGGCGGTATTCGG SEQ ID NO: 66 XhoI ΔG 287- Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC SEQ ID NO: 67 NheI Orf46.1 K Rev CCCGCTCGAG-TTACGTATCATATTTCACGTGC SEQ ID NO: 68 XhoI ΔG 287- Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC SEQ ID NO: 69 NheI 961 K Rev CCCGCTCGAG-TTACCACTCGTAATTGAC SEQ ID NO: 70 XhoI * This primer was used as a Reverse primer for all the 287 forms. ^(§) Forward primers used in combination with the ΔG278 K reverse primer.

Example 6 ORF1 and its Leader Peptide

ORF1 from N. meningitidis (serogroup B, strain MC58) is predicted to be an outer membrane or secreted protein. It has the following sequence (SEQ ID NO:71):

   1 MKTTDKRTTE THRKAPKTGR IRFSPAYLAI CLSFGILPQA WAGHTYFGIN   51 YQYYRDFAEN KGKFAVGAKD IEVYNKKGEL VGKSMTKAPM IDFSVVSRNG  101 VAALVGDQYI VSVAHNGGYN NVDFGAEGRN PDQHRFTYKI VKRNNYKAGT  151 KGHPYGGDYH MPRLHKFVTD AEPVEMTSYM DGRKYIDQNN YPDRVRIGAG  201 RQYWRSDEDE PNERESSYHI ASAYSWLVGG NTFAQNGSGG GTVNLGSEKI  251 KHSPYGFLPT GGSFGDSGSP MFIYDAQKQK WLINGVLQTG NPYIGKSNGF  301 QLVRKDWFYD EIFAGDTHSV FYEPRQNGKY SFNDDNNGTG KINAKHEHNS  351 LPNRLKTRTV QLFNVSLSET AREPVYHAAG GVNSYRPRLN NGENISFIDE  401 GKGELILTSN INQGAGGLYF QGDFTVSPEN NETWQGAGVH ISEDSTVTWK  451 VNGVANDRLS KIGKGTLHVQ AKGENQGSIS VGDGTVILDQ QADDKGKKQA  501 FSEIGLVSGR GTVQLNADNQ FNPDKLYFGF RGGRLDLNGH SLSFERIQNT  551 DEGAMIVNHN QDKESTVTIT GNKDIATTGN NNSLDSKKEI AYNGWFGEKD  601 TTKTNGRLNL VYQPAAEDRT LLLSGGTNLN GNITQTNGKLFFSGRPTPHA  651 YNHLNDHWSQ KEGIPRGEIV WDNDWINRTF KAENFQIKGG QAVVSRNVAK  701 VKGDWHLSNH AQAVFGVAPH QSHTICTRSD WTGLTNCVEK TITDDKVIAS  751 LTKTDISGNV DLADHAHLNL TGLATLNGNL SANGDTRYTV SHNATQNGNL  801 SLVGNAQATF NQATLNGNTS ASGNASFNLS DHAVQNGSLT LSGNAKANVS  851 HSALNGNVSL ADKAVFHFES SRFTGQISGG KDTALHLKDS EWTLPSGTEL  901 GNLNLDNATI TLNSAYRHDA AGAQTGSATD APRRRSRRSR RSLLSVTPPT  951 SVESRFNTLT VNGKLNGQGT FRFMSELFGY RSDKLKLAES SEGTYTLAVN 1001 NTGNEPASLE QLTVVEGKDN KPLSENLNFT LQNEHVDAGA WRYQLIRKDG 1051 EFRLHNPVKE QELSDKLGKA EAKKQAEKDN AQSLDALIAA GRDAVEKTES 1101 VAEPARQAGG ENVGIMQAEE EKKRVQADKD TALAKQREAE TRPATTAFPR 1151 ARRARRDLPQ LQPQPQPQPQ RDLISRYANS GLSEFSATLN SVFAVQDELD 1201 RVFAEDRRNA VWTSGIRDTK HYRSQDFRAY RQQTDLRQIG MQKNLGSGRV 1251 GILFSHNRTE NTFDDGIGNS ARLAHGAVFG QYGIDRFYIG ISAGAGFSSG 1301 SLSDGIGGKI RRRVLHYGIQ ARYRAGFGGF GIEPHIGATR YFVQKADYRY 1351 ENVNIATPGL AFNRYRAGIK ADYSFKPAQH ISITPYLSLS YTDAASGKVR 1401 TRVNTAVLAQ DFGKTRSAEW GVNAEIRGFT LSLHAAAAKG PQLEAQHSAG 1451 IKLGYRW*

The leader peptide is underlined.

A polymorphic form of ORF1 is disclosed in WO99/55 873.

Three expression strategies have been used for ORF1:

-   -   1) ORF1 using a His tag, following WO99/24578 (ORF1-His);     -   2) ORF1 with its own leader peptide but without any fusion         partner (‘ORF1L’); and     -   3) ORF1 with the leader peptide (MKKTAIAIAVALAGFATVAQA (SEQ ID         NO:72)) from E. coli OmpA (‘Orf1LOmpA’) (SEQ ID NO:73):

MKKTAIAIAVALAGFATVAQAASAGHTYFGINYQYYRDFAENKGKFAVGAKDIEVYNKKGELVGKSMTKAPMIDFSV VSRNGVAALVGDQYIVSVAHNGGYNNVDFGAEGRNPDQHRFTYKIVKRNKYKAGTKGHPYGGDYHMPRLHKFVTDAE PVEMTSYMDGRKYIDQNNYPDRVRIGAGRQYWRSDEDEPNNRESSYHIASAYSWLVGGYTFAQNGSGGGTVNLGSEK IKHSPYGFLPTGGSFGDSGSPMFIYDAQKQKWLINGVLQTGNPYIGKSNGFQLVRKDWFYDEIFAGDTHSVFYEPRQ NGKYSFNDDNNGTGKINAKHEHNSLPNRLKTRTVQLFNVSLSETAREFVYHAAGGVNSYRPRLNNGENISFIDEGKG ELILTSNINQGAGGLYFQGDFTVSPENNETWQGAGVHISEDSTVTWKVNGVANDRLSKIGKGTLHVQAKGENQGSIS VGDGTVILDQQADDKGKKQAFSEIGLVSGRGTVQLNADNQFNPDKLYFGFRGGRLDLNGHSLSFHRIQNTDEGAMIV NHNQDKESTVTITGNKDIATTGNNNSLDSKKEIAYNGWFGEKDTTKTNGRLNLVYQPAAEDRTLLLSGGTNLNGNIT QTNGKLFFSGRPTPHAYNHLNDHWSQKEGIPRGEIVWDNDWINRTFRAENFQKGGQAVVSRNVAKVKGDWHLSNHA QAVFGVAPHQSHTICTRSDWTGLTNCVEKTITDDKVIASLTKTDISGNVDLADHAHLNLTGLATLNGNLSANGDTRY TVSHNATQNGNLSLVGNAQATFNQATLNGNTSASGNASFNLSDHAVQNGSLTLSGNAKANVSHSALNGNVSLADKAV FHFESSRPTGQISGGKDTALHLKDSEWTLPSGTELGNLNLDNATITLNSAYRHDAAGAQTGSATDAPRRRSRRSRRS LLSVTPPTSVESRFNTLTVNGKLNGQGTFRFMSELFGYRSDKLKLAESSEGTYTLAVNNTGNEPASLEQLTVVEGKD NKPLSENLNFTLQNEHVDAGAWRYQLIRKDGEFRLHNPVKEQELSDKLGKAEAKKQAEKDNAQSLDALIAAGRDAVE KTESVAEPARQAGGENVGIMQAEEEKKRVQADKDTALAKQREAETRPATTAFPRARRARRDLPQLQPQPQPQPQRDL ISRYANSGLSEFSATLNSVFAVQDELDRVFAEDRRNAVWTSGIRDTKHYRSQDFRAYRQQTDLRQIGMQKNLGSGRV GILFSHNRTENTFDDGIGNSARLAHGAVFGQYGIDRFYIGISAGAGFSSGSLSDGIGGKIRRRVLHYGIQARYRAGF GGFGIEPHIGATRYFVQKADYRYENVNIATPGLAFNRYRAGIKADYSFKPAQHISITPYLSLSYTDAASGKVRTRVN TAVLAQDFGKTRSAEWGVNAEIKGFTLSLHAAAAKGPQLEAQHSAGIKLGYRW*

-   -   To make this construct, the clone pET911LOmpA (see below) was         digested with the NheI and XhoI restriction enzymes and the         fragment corresponding to the vector carrying the OmpA leader         sequence was purified (pETLOmpA). The ORF1 gene coding for the         mature protein was amplified using the oligonucleotides ORF1-For         and ORF1-Rev (including the NheI and XhoI restriction sites,         respectively), digested with NheI and XzoI and ligated to the         purified pETOmpA fragment (see FIG. 1). An additional AS         dipeptide was introduced by the NheI site.

All three forms of the protein were expressed. The His-tagged protein could be purified and was confirmed as surface exposed, and possibly secreted (see FIG. 3). The protein was used to immunise mice, and the resulting sera gave excellent results in the bactericidal assay.

ORF1LOmpA was purified as total membranes, and was localised in both the inner and outer membranes. Unexpectedly, sera raised against ORF1LOmpA show even better ELISA and anti-bactericidal properties than those raised against the His-tagged protein.

ORF1L was purified as outer membranes, where it is localised.

Example 7 Protein 911 and its Leader Peptide

Protein 911 from N. meningitidis (serogroup B, strain MC58) has the following sequence (SEQ ID NO:74):

  1 MKKNILEFWV GLFVLIGAAA VAFLAFRVAG GAAFGGSDKT YAVYADFGDI  51 GGLKVNAPVK SAGVLVGRVG AIGLDPKSYQ ARVRLDLDGK YQFSSDVSAQ 101 ILTSGLLGEQ YIGLQQGGDT ENLAAGDTIS VTSSAMVLEN LIGKFHTSFA 151 EKNADGGNAE KAAE*

The leader peptide is underlined.

Three expression strategies have been used for 911:

-   -   1) 911 with its own leader peptide but without any fusion         partner (‘911L’);     -   2) 911 with the leader peptide from E. coli OmpA (‘911LOmpA’).         -   To make this construct, the entire sequence encoding the             OmpA leader peptide was included in the 5′-primer as a tail             (primer 911LOmpA Forward). A NheI restriction site was             inserted between the sequence coding for the OmpA leader             peptide and the 911 gene encoding the predicted mature             protein (insertion of one amino acid, a serine), to allow             the use of this construct to clone different genes             downstream the OmpA leader peptide sequence.     -   3) 911 with the leader peptide (MKYLLPTAAAGLLLAAQPAMA (SEQ ID         NO:75)) from Erwinia carotovora PelB (‘911LpelB’).     -   To make this construct, the 5′-end PCR primer was designed         downstream from the leader sequence and included the NcoI         restriction site in order to have the 911 fused directly to the         PelB leader sequence; the 3′-end primer included the STOP codon.         The expression vector used was pET22b+ (Novagen), which carries         the coding sequence for the PelB leader peptide. The NcoI site         introduces an additional methionine after the PelB sequence.

All three forms of the protein were expressed. ELISA titres were highest using 911L, with 919LOmpA also giving good results.

Example 8 ORF46

The complete ORF46 protein from N. meningitidis (serogroup B, strain 2996) has the following sequence (SEQ ID NO:76):

  1 LGISRKISLI LSILAVCLPM HAHASDLAND SFIRQVLDRQ HFEPDGKYHL  51 FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY IVRFSDHGHE 101 VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY 151 PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH NAGSMLTQGV 201 GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE IVGAGDAVQG 251 ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA AIRDWAVQNP 301 NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP 351 KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI TSSTVPPSNM 401 KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTKLDIQEL SGGGIPKAKP 451 VSDAKPRWEV DRKLNKLTTR EQVEKNVQEI RNGNKNSNFS QHAQLEREIN 501 KLKSADEINF ADGMGKFTDS MNDKAFSRLV KSVKENGFTN PVVEYVEING 551 KAYIVRGNNR VFAAEYLGRI HELKFKKVDF PVPNTSWKNP TDVLNESGNV 601 KRPRYRSK*

The leader peptide is underlined.

The sequences of ORF46 from other strains can be found in WO00/66741.

Three expression strategies have been used for ORF46:

-   -   1) ORF46 with its own leader peptide but without any fusion         partner (‘ORF46-2L’);     -   2) ORF46 without its leader peptide and without any fusion         partner (‘ORF46-2’), with the leader peptide omitted by         designing the 5′-end amplification primer downstream from the         predicted leader sequence (SEQ ID NO:77):

  1 SDLANDSFIR QVLDRQHFEP DGKYHLFGSR GELAERSGHI GLGKIQSHQL  51 GNLMIQQAAI KGNIGYIVRF SDHGHEVHSP FDNHASHSDS DEAGSPVDGF 101 SLYRIHWDGY EHHPADGYDG PQGGGYPAPK GARDIYSYDI KGVAQNIRLN 151 LTDNRSTGQR LADRFHNAGS MLTQGVGDGF KRATRYSPEL DRSGNAAEAF 201 NGTADIVKNI IGAAGEIVGA GDAVQGISEG SNIAVMHGLG LLSTENKMAR 251 INDLADMAQL KDYAAAAIRD WAVQNPNAAQ GIEAVSNIFM AAIPIKGIGA 301 VRGKYGLGGI TAHPIKRSQM GAIALPKGKS AVSDNFADAA YAKYPSPYHS 351 RNIRSNLEQR YGKENITSST VPPSNGKNVK LADQRHPKTG VPFDGKGFPN 401 FEKHVKYDTK LDIQELSGGG IPKAKPVSDA KPRWEVDRKL NKLTTREQVE 451 KNVQEIRNGN KNSNFSQHAQ LEREINKLKS ADEINFADGM GKFTDSMNDK 501 AFSRLVKSVK ENGFTNPVVE YVEINGKAYI VRGNNRVFAA EYLGRIHELK 551 FKKVDFPVPN TSWKNPTDVL NESGNVKRPR YRSK*

-   -   3) ORF46 as a truncated protein, consisting of the first 433         amino acids (‘ORF46.1L’), constructed by designing PCR primers         to amplify a partial sequence corresponding to aa 1-433.     -   A STOP codon was included in the 3′-end primer sequences.

ORF46-2L is expressed at a very low level to E. coli. Removal of its leader peptide (ORF46-2) does not solve this problem. The truncated ORF46.1L form (first 423 amino acids, which are well conserved between serogroups and species), however, is well-expressed and gives excellent results in ELISA test and in the bactericidal assay.

ORF46.1 has also been used as the basis of hybrid proteins. It has been fused with 287, 919, and ORF1. The hybrid proteins were generally insoluble, but gave some good ELISA and bactericidal results (against the homologous 2996 strain):

Protein ELISA Bactericidal Ab Orf1-Orf46.1-His  850  256 919-Orf46.1-His 12900  512 919-287-Orf46-His n.d. n.d. Orf46.1-287His  150  8192 Orf46.1-919His  2800  2048 Orf46.1-287-919His  3200 16384

For comparison, ‘triple’ hybrids of ORF46.1, 287 (either as a GST fusion, or in ΔG287 form) and 919 were constructed and tested against various strains (including the homologous 2996 strain) versus a simple mixture of the three antigens. FCA was used as adjuvant:

2996 BZ232 MC58 NGH38 F6124 BZ133 Mixture 8192 256 512 1024 >2048 >2048 ORF46.1-287- 16384 256 4096 8192 8192 8192 919his ΔG287-919- 8192 64 4096 8192 8192 16384 ORF46.1his ΔG287-ORF46.1- 4096 128 256 8192 512 1024 919his

Again, the hybrids show equivalent or superior immunological activity.

Hybrids of two proteins (strain 2996) were compared to the individual proteins against various heterologous strains:

1000 MC58 F6124 (MenA) ORF46.1-His <4   4096  <4   ORF1-His 8 256 128  ORF1-ORF46.1-His 1024   512 1024  

Again, the hybrid shows equivalent or superior immunological activity.

Example 9 Protein 961

The complete 961 protein from N. meningitidis (serogroup B, strain MC58) has the following sequence (SEQ ID NO:78):

  1 MSMKHFPAKV LTTAILATFC SGALAATSDD DVKKAATVAI VAAYNNGQEI  51 NGFKAGETIY DIGEDGTITQ KDATAADVEA DDFKGLGLKK VVTNLTKTVN 101 ENKQNVDAKV KAAESEIEKL TTKLADTDAA LADTDAALDE TTNALNKLGE 151 NITTFAEETK TNIVKIDEKL EAVADTVDKH AEAFNDIADS LDETNTKADE 201 AVKTANEAKQ TAEETKQNVD AKVKAAETAA GKAEAAAGTA NTAADKAEAV 251 AAKVTDIKAD IATNKADIAK NSARIDSLDK NVANLRKETR QGLAEQAALS 301 GLFQPYNVGR FNVTAAVGGY KSESAVAIGT GFRFTENFAA KAGVAVGTSS 351 GSSANYHVGV NYEW*

The leader peptide is underlined.

Three approaches to 961 expression were used:

-   -   1) 961 using a GST fusion, following WO99/572810 (‘GST961’);     -   2) 961 with its own leader peptide but without any fusion         partner (‘961L’); and     -   3) 961 without its leader peptide and without any fusion         partner. (‘961^(untagged)’), with the leader peptide omitted by         designing the 5′-end PCR primer downstream from the predicted         leader sequence.

All three forms of the protein were expressed. The GST-fusion protein could be purified and antibodies against it confirmed that 961 is surface exposed (FIG. 4). The protein was used to immunise mice, and the resulting sera gave excellent results in the bactericidal assay. 961L could also be purified and gave very high ELISA titres.

Protein 961 appears to be phase variable. Furthermore, it is not found in all strains of N. meningitidis.

Example 10 Protein 287

Protein 287 from N. meningitidis (serogroup B, strain 2996) has the following sequence (SEQ ID NO:79):

  1 MFERSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP VVAEKETEVK  51 EDAPQAGSQG QGAPSTQGSQ DMAAVSAENT GNGGAATTDK PKNEDEGPQN 101 DMPQNSAESA NQTGNNQPAD SSDSAPASNP APANGGSNFG RVDLANGVLI 151 DGPSQNITLT HCKGDSCNGD NLLDEEAPSK SEFENLNESE RIEKYKKDGK 201 SDKFTNLVAT AVQANGTNKY VIIYKDKSAS SSSARFRRSA RSRRSLPAEM 251 PLIPVNQADT LIVDGEAVSL TGHSGNIFAP EGNYRYLTYG AEKLPGGSYA 301 LRVQGEPAKG EMLAGTAVYN GEVLHFHTEN GRPYPTRGRF AAKVDFGSKS 351 VDGIIDSGDD LHMGTQKFKA AIDGNGFKGT WTENGGGDVS GRFYGPAGEE 401 VAGKYSYRPT DAEKGGFGVF AGKKEQD*

The leader peptide is shown underlined.

The sequences of 287 from other strains can be found in FIGS. 5 and 15 of WO00/66741.

Example 9 of WO99/57280 discloses the expression of 287 as a GST-fusion in E. coli.

A number of further approaches to expressing 287 in E. coli have been used, including:

-   -   1) 287 as a His-tagged fusion (‘287-His’);     -   2) 287 with its own leader peptide but without any fusion         partner (‘287L’);     -   3) 287 with the ORF4 leader peptide and without any fusion         partner (‘287LOrf4’); and     -   4) 287 without its leader peptide and without any fusion partner         (‘287^(untagged)’) (SEQ ID NO:80):

  1 CGGGGGGSPD VKSADTLSKP AAPVVAEKET EVKEDAPQAD SQGQGAPSTQ  51 GSQDMAAVSA ENTGNGGAAT TDKPKNEDEG PQNDMPQNSA ESANQTGNNQ 101 PADSSDSAPA SNPAPANGGS NFGRVDLANG VLIDGPSQNI TLTHCKGDSC 151 NGDNLLDEEA PSKSEFENLN ESERIEKYKK DGKSDKFTNL VATAVQANGT 201 NKYVIIYKDK SASSSSARFR RSARSRRSLP AEMPLIPVNQ ADTLIVDGEA 251 VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP AKGEMLAGTA 301 VYNGEVLHFH TENGRPYPTR GRFAAKVDFG SKSVDGIIDS GDDLHMGPQK 351 FKAAIDGNGF KGTWTENGGG DVSGRFYGPA GEEVAGKYSY RPTDAEKGGF 401 GVFAGKKEQD *

All these proteins could be expressed and purified.

‘287L’ and ‘287LOrf4’ were confirmed as lipoproteins.

As shown in FIG. 2, ‘287LOrf4’ was constructed by digesting 919LOrf4 with NheI and XhoI. The entire ORF4 leader peptide was restored by the addition of a DNA sequence coding for the missing amino acids, as a tail, in the 5′-end primer (287LOrf4 for), fused to 287 coding sequence. The 287 gene coding for the mature protein was amplified using the oligonucleotides 287LOrf4 For and Rev (including the NheI and XhoI sites, respectively), digested with NheI and XhoI and ligated to the purified pBTOrf4 fragment.

Example 11 Further Non-Fusion Proteins with/without Native Leader Peptides

A similar approach was adopted for E. coli expression of further proteins from WO99/24578, WO99/36544 and WO99/57280.

The following were expressed without a fusion partner: 008, 105, 117-1, 121-1, 122-1, 128-1, 148, 216, 243, 308, 593, 652, 726, 982, and Orf143-1. Protein 117-1 was confirmed as surface-exposed by FACS and gave high ELISA titres.

The following were expressed with the native leader peptide but without a fusion partner: 111, 149, 206, 225-1, 235, 247-1, 274, 283, 286, 292, 401, 406, 502-1, 503, 519-1, 525-1, 552, 556, 557, 570, 576-1, 580, 583, 664, 759, 907, 913, 920-1, 926, 936-1, 953, 961, 983, 989, Orf4, Orf7-1, Orf9-1, Orf23, Orf25, Orf37, Orf38, Orf40, Orf40.1, Orf40.2, Orf72-1, Orf76-1, Orf85-2, Orf91, Orf97-1, Orf119, Orf143.1. These proteins are given the suffix ‘L’.

His-tagged protein 760 was expressed with and without its leader peptide. The deletion of the signal peptide greatly increased expression levels. The protein could be purified most easily using 2M urea for solubilisation.

His-tagged protein 264 was well-expressed using its own signal peptide, and the 30 kDa protein gave positive Western blot results.

All proteins were successfully expressed.

The localisation of 593, 121-1, 128-1, 593, 726, and 982 in the cytoplasm was confirmed.

The localisation of 920-1L, 953L, ORF9-1L, ORF85-2L, ORF97-1L, 570L, 580L and 664L in the periplasm was confirmed.

The localisation of ORF40L in the outer membrane, and 008 and 519-1L in the inner membrane was confirmed. ORF25L, ORF4L, 406L, 576-1L were all confirmed as being localised in the membrane.

Protein 206 was found not to be a lipoprotein.

ORF25 and ORF40 expressed with their native leader peptides but without fusion partners, and protein 593 expressed without its native leader peptide and without a fusion partner, raised good anti-bactericidal sera. Surprisingly, the forms of ORF25 and ORF40 expressed without fusion partners and using their own leader peptides (i.e. ‘ORF25L’ and ‘ORF40L’) give better results in the bactericidal assay than the fusion proteins.

Proteins 920L and 953L were subjected to N-terminal sequencing, giving HRVWVETAH (SEQ ID NO:81) and ATYKVDEYHANARFAF (SEQ ID NO:82), respectively. This sequencing confirms that the predicted leader peptides were cleaved and, when combined with the periplasmic location, confirms that the proteins are correctly processed and localised by E. coli when expressed from their native leader peptides.

The N-terminal sequence of protein 519.1L localised in the inner membrane was MEFFIILLA (SEQ ID NO:83), indicating that the leader sequence is not cleaved. It may therefore function as both an uncleaved leader sequence and a transmembrane anchor in a manner similar to the leader peptide of PBP1 from N. gonorrhoeae [Ropp & Nicholas (1997) J. Bact. 179:2783-2787.].Indeed the N-terminal region exhibits strong hydrophobic character and is predicted by the Tmpred. program to be transmembrane.

Example 12 Lipoproteins

The incorporation of palmitate in recombinant lipoproteins was demonstrated by the method of Kraft et. al. [J. Bact. (1998) 180:3441-3447.]. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. The culture was diluted to an OD₅₅₀ of 0.1 in 5.0 ml of fresh medium LB/Amp medium containing 5 μC/ml [³H] palmitate (Amersham). When the OD₅₅₀ of the culture reached 0.4-0.8, recombinant lipoprotein was induced for 1 hour with IPTG (final concentration 1.0 mM). Bacteria were harvested by centrifugation in a bench top centrifuge at 2700 g for 15 min and washed twice with 1.0 ml cold PBS. Cells were resuspended in 120 μl of 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1.0% w/v SDS and lysed by boiling for 10 min. After centrifugation at 13000 g for 10 min the supernatant was collected and proteins precipitated by the addition of 1.2 ml cold acetone and left for 1 hour at −20° C. Protein was pelleted by centrifugation at 13000 g for 10 min and resuspended in 20-50 μl (calculated to standardise loading with respect to the final O.D of the culture) of 1.0% w/v SDS. An aliquot of 15 μl was boiled with 5 μl of SDS-PAGE sample buffer and analysed by SDS-PAGE. After electrophoresis gels were fixed for 1 hour in 10% v/v acetic acid and soaked for 30 minutes in Amplify solution (Amersham). The gel was vacuum-dried under heat and exposed to Hyperfilm (Kodak) overnight −80° C.

Incorporation of the [³H] palmitate label, confirming lipidation, was found for the following proteins: Orf4L, Orf25L, 287L, 287LOrf4, 406.L, 576L, 926L, 919L and 919LOrf4.

Example 13 Domains in 287

Based on homology of different regions of 287 to proteins that belong to different functional classes, it was split into three ‘domains’, as shown in FIG. 5. The second domain shows homology to IgA proteases, and the third domain shows homology to transferrin-binding proteins.

Each of the three ‘domains’ shows a different degree of sequence conservation between N. meningitidis strains—domain C is 98% identical, domain A is 83% identical, whilst domain B is only 71% identical. Note that protein 287 in strain MC58 is 61 amino acids longer than that of strain 2996. An alignment of the two sequences is shown in FIG. 7, and alignments for various strains are disclosed in WO00/66741 (see FIGS. 5 and 15 therein).

The three domains were expressed individually as C-terminal His-tagged proteins. This was done for the MC58 and 2996 strains, using the following constructs:

-   -   287a-MC58 (aa 1-202), 287b-MC58 (aa 203-288), 287c-MC58 (aa         311-488).     -   287a-2996 (aa 1-139), 287b-2996 (aa 140-225), 287c-2996 (aa         250-427).

To make these constructs, the stop codon sequence was omitted in the 3′-end primer sequence. The 5′ primers included the NheI restriction site, and the 3′ primers included a XhoI as a tail, in order to direct the cloning of each amplified fragment into the expression vector pET21b+ using NdeI-XhoI, NheI-XhoI, or NdeI-HindIII restriction sites.

All six constructs could be expressed, but 287b-MC8 required denaturation and refolding for solubilisation.

Deletion of domain A is described below (‘Δ4 287-His’).

Immunological data (serum bactericidal assay) were also obtained using the various domains from strain 2996, against the homologous and heterologous MenB strains, as well as MenA (F6124 strain) and MenC (BZ133 strain):

2996 BZ232 MC58 NGH38 394/98 MenA MenC   287-His 32000 16 4096 4096 512 8000 16000 287(B)-His 256 — — — — 16 — 287(C)-His 256 — 32 512 32 2048 >2048 287(B-C)-His  64000 128 4096 64000 1024 64000 32000

Using the domains of strain MC58, the following results were obtained:

MC58 2996 BZ232 NGH38 394/98 MenA MenC    287-His 4096 32000 16 4096 512 8000 16000  287(B)-His 128 128 — — — — 128  287(C)-His — 16 — 1024 — 512 — 287(B-C)-His 16000 64000 128 64000 512 64000 >8000

Example 14 Deletions in 287

As well as expressing individual domains, 287 was also expressed (as a C-terminal His-tagged protein) by making progressive deletions within the first domain. These

Four deletion mutants of protein 287 from strain 2996 were used (FIG. 6):

-   -   1) ‘287-His’, consisting of amino acids 18-427 (i.e. leader         peptide deleted);     -   2) ‘Δ1 287-His’, consisting of amino acids 26-427;     -   3) ‘Δ2 287-His’, consisting of amino acids 70-427;     -   4) ‘Δ3 287-His’, consisting of amino acids 107-427; and     -   5) ‘Δ4 287-His’, consisting of amino acids 140-427 (=287-bc).

The ‘Δ4’ protein was also made for strain MC58 (‘Δ4 287MC58-His’; aa 203-488).

The constructs were made in the same way as 287a/b/c, as described above.

All six constructs could be expressed and protein could be purified. Expression of 287-His was, however, quite poor.

Expression was also high when the C-terminal His-tags were omitted.

Immunological data (serum bactericidal assay) were also obtained using the deletion mutants, against the homologous (2996) and heterologous MenB strains, as well as MenA (F6124 strain) and MenC (BZ133 strain):

2996 BZ232 MC58 NGH38 394/98 MenA MenC   287-his 32000 16 4096 4096 512 8000 16000 Δ1 287-His 16000 128 4096 4096 1024 8000 16000 Δ2 287-His 16000 128 4096 >2048 512 16000 >8000 Δ3 287-His 16000 128 4096 >2048 512 16000 >8000 Δ4 287-His 64000 128 4096 64000 1024 64000 32000

The same high activity for the Δ4 deletion was seen using the sequence from strain MC58.

As well as showing superior expression characteristics, therefore, the mutants are immunologically equivalent or superior.

Example 15 Poly-Glycine Deletions

The ‘Δ1 287-His’ construct of the previous example differs from 287-His and from ‘287^(untagged)’ only by a short N-terminal deletion (GGGGGGS) (SEQ ID NO:631). Using an expression vector which replaces the deleted serine with a codon present in the Nhe cloning site, however, this amounts to a deletion only of (Gly)₆(SEQ ID NO:632). Thus, the deletion of this (Gly)₆ sequence (SEQ ID NO:632) has been shown to have a dramatic effect on protein expression.

The protein lacking the N-terminal amino acids up to GGGGGG (SEQ ID NO: 632) is called ‘ΔG 287’. In strain MC58, its sequence (leader peptide underlined) is (SEQ ID NO: 84):

 ΔG287 1 MFKRSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP VVSEKETEAK 51 EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT GNGGAVTADN PKNEDEVAQN 101 DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP ANQPDMANAA 151 DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA SNPAPANGGS 201 NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS 251 DADKISNYKK DGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSFARFRRS 301 ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS LTGHSGNIFA PEGNYRYLTY 351 GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY NGEVLHFHTE NGRPYPTRGR 401 FAAKVDFGSK SVDGIIDSGD DLHMGTQKFK AAIDGNGFKG TWTENGSGDV 451 SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD*

ΔG287, with or without His-tag (‘ΔG287-His’ and ‘ΔG287K’, respectively), are expressed at very good levels in comparison with the ‘287-His’ or ‘287^(untagged)’.

On the basis of gene variability data, variants of ΔG287-His were expressed in E. coli from a number of MenB strains, in particular from strains 2996, MC58, 1000, and BZ232. The results were also good.

It was hypothesised that poly-Gly deletion might be a general strategy to improve expression. Other MenB lipoproteins containing similar (Gly)_(n) motifs (near the N-terminus, downstream of a cysteine) were therefore identified, namely Tbp2 (NMB0460) (SEQ ID NO:85), 741 (NMB 1870) (SEQ ID NO:86) and 983 (NMB1969) (SEQ ID NO:87):

TBP2 

 ΔGTbp2 1 MNNPLVNQAA MVLPVFLLSA CLGGGGSFDL DSVDTEAPRP APKYQDVFSE 51 KPQAQKDQGG YGFAMRLKRR NWYPQAKEDE VKLDESDWEA TGLPDEPKEL 101 PKRQKSVIEK VETDSDNNIY SSPYLKPSNH QNGNTGNGIN QPKNQAKDYE 151 NFKYVYSGWF YKHAKREFNL KVEPKSAKNG DDGYIFYHGK EPSRQLPASG 201 KITYKGVWHF ATDTKKGQKF REIIQPSKSQ GDRYSGFSGD DGEEYSNKNK 251 STLTDGQEGY GFTSNLEVDF HNKKLTGKLI RNNANTDNNQ ATTTQYYSLE 301 AQVTGNRFNG KATATDKPQQ NSETKEHPFV SDSSSLSGGF FGPQGEELGF 351 RFLSDDQKVA VVGSAKTKDK PANGNTAAAS GGTDAAASNG AAGTSSENGK 401 LTTVLDAVEL KLGDKEVQKL DNFSNAAQLV VDGIMIPLLP EASESGNNQA 451 NQGTNGGTAF TRKFDHTPES DKKDAQAGTQ TNGAQTASNT AGDTNGKTKT 501 YEVEVCCSNL NYLKYGMLTR KNSKSAMQAG ESSSQADAKT EQVEQSMFLQ 551 GERTDEKEIP SEQNIVYRGS WYGYIANDKS TSWSGNASNA TSGNRAEFTV 601 NFADKKITGT LTADNRQEAT FTIDGNIKDN GFEGTAKTAE SGFDLDQSNT 651 TRTPKAYITD AKVQGGFYGP KAEELGGWFA YPGDKQTKNA TNASGNSSAT 701 VVFGAKRQQP VR* 741 

 ΔG741 1 VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL 51 QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ 101 IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI 151 GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI 201 EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA 251 QEVAGSAEVK TVNGIRHIGL AAKQ* 983 

 ΔG983 1 MRTTPTFPTK TFKPTAMALA VATTLSACLG GGGGGTSAPD FNAGGTGIGS 51 NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN 101 LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE 151 LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT 201 DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIN NTNDETKNEM 251 MVAAIRNAWV KLGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL 301 DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NNLFIFSTGN DAQAQPNTYA 351 LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA 401 MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN 451 LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT 501 SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN 551 KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ 601 LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS 651 AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY YVRRGNAART 701 ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT 751 DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQG 801 RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMR GSTQTVGIAA 851 KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL 901 FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE 951 GGLRYDLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL 1001 FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE 1051 FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*

Tbp2 and 741 genes were from strain MC58; 983 and 287 genes were from strain 2996. These were cloned in pET vector and expressed in E. coli without the sequence coding for their leader peptides or as “ΔG forms”, both fused to a C-terminal His-tag. In each case, the same effect was seen—expression was good in the clones carrying the deletion of the poly-glycine stretch, and poor or absent if the glycines were present in the expressed protein:

ORF Express. Purification Bact. Activity 287-His(2996) +/− + + ‘287^(untagged)’(2996) +/− nd nd ΔG287-His(2996) + + + ΔG287K(2996) + + + ΔG287-His(MC58) + + + ΔG287-His(1000) + + + ΔG287-His(BZ232) + + + Tbp2-His(MC58) +/− nd nd ΔGTbp2-His(MC58) + + 741-His(MC58) +/− nd nd ΔG741-His(MC58) + + 983-His (2996) ΔG983-His (2996) + +

SDS-PAGE of the proteins is shown in FIG. 13.

ΔG287 and Hybrids

ΔG287 proteins were made and purified for strains MC58, 1000 and BZ232. Each of these gave high ELISA titres and also serum bactericidal titres of >8192. ΔG287K, expressed from pET-24b, gave excellent titres in ELISA and the serum bactericidal assay. ΔG287-ORF46.1K may also be expressed in pET-24b.

ΔG287 was also fused directly in-frame upstream of 919(SEQ ID NOS:88 and 89), 953 (SEQ ID NOS:90 and 91), 961 (SEQ ID NOS:92 and 93) (sequences shown below) and ORF46.1:

ΔG287-919 1 ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC 51 TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG 101 CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCAG CCAAGATATG 151 GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA 201 CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT 251 CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA 301 GATTCCGCCC CCGCGTCAAA CCCTGCACCT GCGAATGGCG GTAGCAATTT 351 TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA 401 ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA 451 TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC 501 TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA 551 ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC 601 ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC 651 TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA 701 ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG 751 CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA 801 CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG 851 AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA 901 GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG 951 GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG 1001 ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC 1051 GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT 1101 TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACh 1151 GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC 1201 AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGATGCCAAA GCAAGAGCAT 1251 CCAAACCTTT CCGCAACCCG ACACATCCGT CATCAACGGC CCGGACCGGC 1301 CGGTCGGCAT CCCCGACCCC GCCGGAACGA CGGTCGGCGG CGGCGGGGCC 1351 GTCTATACCG TTGTACCGCA CCTGTCCCTG CCCCACTGGG CGGCGCAGGA 1401 TTTCGCCAAA AGCCTGCAAT CCTTCCGCCT CGGCTGCGCC AATTTGAAAA 1451 ACCGCCAAGG CTGGCAGGAT GTGTGCGCCC AAGCCTTTCA AACCCCCGTC 1501 CATTCCTTTC AGGCAAAACA GTTTTTTGAA CGCTATTTCA CGCCGTGGCA 1551 GGTTGCAGGC AACGGAAGCC TTGCCGGTAC GGTTACCGGC TATTACGAGC 1601 CGGTGCTGAA GGGCGACGAC AGGCGGACGG CACAAGCCCG CTTCCCGATT 1651 TACGGTATTC CCGACGATTT TATCTCCGTC CCCCTGCCTG CCGGTTTGCG 1701 GAGCGGAAAA GCCCTTGTCC GCATCAGGCA GACGGGAAAA AACAGCGGCA 1751 CAATCGACAA TACCGGCGGC ACACATACCG CCGACCTCTC CCGATTCCCC 1801 ATCACCGCGC GCACAACGGC AATCAAAGGC AGGTTTGAAG GAAGCCGCTT 1851 CCTCCCCTAC CACACGCGCA ACCAAATCAA CGGCGGCGCG CTTGACGGCA 1901 AAGCCCCGAT ACTCGGTTAC GCCGAAGACC CCGTCGAACT TTTTTTTATG 1951 CACATCCAAG GCTCGGGCCG TCTGAAAACC CCGTCCGGCA AATACATCCG 2001 CATCGGCTAT GCCGACAAAA ACGAACATCC CTACGTTTCC ATCGGACGCT 2051 ATATGGCGGA CAAAGGCTAC CTCAAGCTCG GGCACACCTC GATGCAGGGC 2101 ATCAAAGCCT ATATGCGGCA AAATCCGCAA CGCCTCGCCG AAGTTTTGGG 2151 TCAAAACCCC AGCTATATCT TTTTCCGCGA GCTTGCCGGA AGCAGCAATG 2201 ACGGTCCCGT CGGCGCACTG GGCACGCCGT TGATGGGGGA ATATGCCGGC 2251 GCAGTCGACC GGCACTACAT TACCTTGGGC GCGCCCTTAT TTGTCGCCAC 2301 CGCCCATCCG GTTACCCGCA AAGCCCTCAA CCGCCTGATT ATGGCGCAGG 2351 ATACCGGCAG CGCGATTAAA GGCGCGGTGC GCGTGGATTA TTTTTGGGGA 2401 TACGGCGACG AAGCCGGCGA ACTTGCCGGC AAACAGAAAA CCACGGGTTA 2451 CGTCTGGCAG CTCCTACCCA ACGGTATGAA GCCCGAATAC CGCCCGTAAC 2501 TCGAG 1 MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM 51 AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS 101 DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL 151 LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI 201 IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG 251 HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE 301 VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI 351 DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG 401 KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA 451 VYTVVPHLSL PHWAAQDFAK SLQSFRLGCA NLKNRQGWQD VCAQAPQTPV 501 HSFQAKQFFE RYFTPWQVAG NGSLAGTVTG YYEPVLKGDD RRTAQARFPI 551 YGIPDDFISV PLPAGLRSGK ALVRIRQTGK NSGTIDNTGG THTADLSRFP 601 ITARTTAIKG RFEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM 651 HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LKLGQTSMQG 701 IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG 751 AVDRHYITLG APLFVATAHP VTRKALNRLI MAQDTGSAIK GAVRVDYFWG 801 YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP* ΔG287-953 1 ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC 51 TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG 101 CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGCCCG CCAAGATATG 151 GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA 201 CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT 251 CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA 301 GATTCCGCCC CCGCGTCAAA CCCTGCACCT GCGAATGGCG GTAGCAATTT 351 TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA 401 ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA 451 TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC 501 TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA 551 ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC 601 ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC 651 TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA 701 ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG 751 CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA 801 CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG 851 AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA 901 GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG 951 GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG 1001 ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC 1051 GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT 1101 TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA 1151 GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC 1201 AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGAGCCACCT ACAAAGTGGA 1251 CGAATATCAC GCCAACGCCC GTTTCGCCAT CGACCATTTC AACACCAGCA 1301 CCAACGTCGG CGGTTTTTAC GGTCTGACCG GTTCCGTCGA GTTCGACCAA 1351 GCAAAACGCG ACGGTAAAAT CGACATCACC ATCCCCGTTG CCAACCTGCA 1401 AAGCGGTCAG CAACACTTTA CCGACCACCT GAAATCAGCC GGCATTATCG 1451 ATGCCGCCCA ATATCCGGAC ATCCGCTTTG TTTCCACCAA ATTCAACTTC 1501 AACGGCAAAA AACTGGTTTC CGTTGACGGC AACCTGACCA TGCACGGCAA 1551 AACCGCCCCC GTCAAACTCA AAGCCGAAAA ATTCAACTGC TACCAAAGCC 1601 CGATGGCGAA AACCGAAGTT TGCGGCGGCG ACTTCAGCAC CACCATCGAC 1651 CGCACCAAAT GGGGCGTGGA CTACCTCGTT AACGTTGGTA TGACCAAAAG 1701 CGTCCGCATC GACATCCAAA TCGAGGCAGC CAAACAATAA CTCGAG 1 MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM 51 AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS 101 DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL 151 LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI 201 IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG 251 HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE 301 VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI 351 DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG 401 KKEQDGSGGG GATYKVDEYH ANARFAIDHF NTSTNVGGFY GLTGSVEFDQ 451 AKRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTKFNF 501 NGKKLVSVDG NLTMHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID 551 RTKWGVDYLV NVGMTKSVRI DIQIEAAKQ* ΔG287-961 1 ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC 51 TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG 101 CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCAG CCAAGATATG 151 GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA 201 CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT 251 CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA 301 GATTCCGCCC CCGCGTCAAA CCCTGCACCT GCGAATGGCG GTAGCAATTT 351 TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGCGTCAAA 401 ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA 451 TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC 501 TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA 551 ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC 601 ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC 651 TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA 701 ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG 751 CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA 801 CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG 851 AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA 901 GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG 951 GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG 1001 ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC 1051 GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT 1101 TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA 1151 GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC 1201 AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGAGCCACAA ACGACGACGA 1251 TGTTAAAAAA GCTGCCACTG TGGCCATTGC TGCTGCCTAC AACAATGGCC 1301 AAAAATTCAA CGGTTTCAAA GCTGGAGAGA CCATCTACGA CATTGATGAA 1351 GACGGCACAA TTACCAAAAA AGACGCAACT GCAGCCGATG TTGAAGCCGA 1401 CGACTTTAAA GGTCTGGGTC TGAAAAAAGT CGTGACTAAC CTGACCAAAA 1451 CCGTCAATGA AAACAAACAA AACGTCGATG CCAAAGTAAA AGCTGCAGAA 1501 TCTGAAATAG AAAAGTTAAC AACCAAGTTA GCAGACACTG ATGCCGCTTT 1551 AGCAGATACT GATGCCGCTC TGGATGCAAC CACCAACGCC TTGAATAAAT 1601 TGGGAGAAAA TATAACGACA TTTGCTGAAG AGACTAAGAC AAATATCGTA 1651 AAAATTGATG AAAAATTAGA AGCCGTGGCT GATACCGTCG ACAAGCATGC 1701 CGAAGCATTC AACGATATCG CCGATTCATT GGATGAAACC AACACTAAGG 1751 CAGACGAAGC CGTCAAAACC GCCAATGAAG CCAAACAGAC GGCCGAAGAA 1801 ACCAAACAAA ACGTCGATGC CAAAGTAAAA GCTGCAGAAA CTGCAGCAGG 1851 CAAAGCCGAA GCTGCCGCTG GCACAGCTAA TACTGCAGCC GACAAGGCCG 1901 AAGCTGTCGC TGCLAAAGTT ACCGACATCA AAGCTGATAT CGCTACGAAC 1951 AAAGATAATA TTGCTAAAAA AGCAAACAGT GCCGACGTGT ACACCAGAGA 2001 AGAGTCTGAC AGCAAATTTG TCAGAATTGA TGGTCTGAAC GCTACTACCG 2051 AAAAATTGGA CACACGCTTG GCTGCCGCTG AAAAATCCAT TGCCGATCAC 2101 GATACTCGCC TGAACGGTTT GGATAAAACA GTGTCAGACC TGCGCAAAGA 2151 AACCCGCCAA GGCCTTGCAG AACAAGCCGC GCTCTCCGGT CTGTTCCAAC 2201 CTTACAACGT GGGTCGGTTC AATGTAACGG CTGCAGTCGG CGGCTACAAA 2251 TCCGAATCGG CAGTCGCCAT CGGTACCGGC TTCCGCTTTA CCGAAAACTT 2301 TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC TTCGTCCGGT TCTTCCGCAG 2351 CCTACCATGT CGGCGTCAAT TACGAGTGGT AACTCGAG 1 MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM 51 AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS 101 DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL 151 LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI 201 IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG 251 HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE 301 VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI 351 DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG 401 KKEQDGSGGG GATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE 451 DGTITKKDAT AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE 501 SEIEKLTTKL ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV 551 KIDEKLEAVA DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE 601 TKQNVDAKVK AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN 651 KDNIAKKANS ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH 701 DTRLNGLDKT VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK 751 SESAVAIGTG FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEW*

ELISA Bactericidal ΔG287-953-His 3834 65536 ΔG287-961-His 108627 65536

The bactericidal. efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens (using 287-GST) for 919 and ORF46.1:

Mixture with 287 Hybrid with ΔG287 919 32000 128000 ORF46.1 128 16000

Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained:

919 ORF46.1 Strain Mixture Hybrid Mixture Hybrid NGH38 1024 32000 — 16384  MC58 512 8192 —  512 BZ232 512 512 — — MenA (F6124) 512 32000 — 8192 MenC (C11) >2048 >2048 — — MenC (BZ133) >4096 64000 — 8192

The hybrid proteins with ΔG287 at the N-terminus are therefore immunologically superior to simple mixtures, with ΔG287-ORF46.1 being particularly effective, even against heterologous strains. ΔG287-ORF46.1K may be expressed in pET-24b.

The same hybrid proteins were made using New Zealand strain 394/98 rather than 2996:

ΔG287NZ-919 (SEQ ID NOS: 94 and 95) 1 ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC 51 CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG 101 CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG 151 GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA 201 CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG 251 CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG 301 CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGCCGGGG AATCGGAGCA 351 GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG 401 ACGATCCGTC GGCAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT 451 ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC 501 TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA 551 ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG 601 ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA 651 AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA 701 GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC 751 GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT 801 CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC 851 GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG 901 GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC 951 CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG 1001 CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT 1051 TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT 1101 GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG 1151 CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC 1201 GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG 1251 AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG 1301 GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT 1351 CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA 1401 AGAGCAGGAT GGATCCGGAG GAGGAGGATG CCAAAGCAAG AGCATCCAAA 1451 CCTTTCCGCA ACCCGACACA TCCGTCATCA ACGGCCCGGA CCGGCCGGTC 1501 GGCATCCCCG ACCCCGCCGG AACGACGGTC GGCGGCGGCG GGGCCGTCTA 1551 TACCGTTGTA CCGCACCTGT CCCTGCCCCA CTGGGCGGCG CAGGATTTCG 1601 CCAAAAGCCT GCAATCCTTC CGCCTCGGCT GCGCCAATTT GAAAAACCGC 1651 CAAGGCTGGC AGGATGTGTG CGCCCAAGCC TTTCAAACCC CCGTCCATTC 1701 CTTTCAGGCA AAACAGTTTT TTGAACGCTA TTTCACGCCG TGGCAGGTTG 1751 CAGGCAACGG AAGCCTTGCC GGTACGGTTA CCGGCTATTA CGAGCCGGTG 1801 CTGAAGGGCG ACGACAGGCG GACGGCACAA GCCCGCTTCC CGATTTACGG 1851 TATTCCCGAC GATTTTATCT CCGTCCCCCT GCCTGCCGGT TTGCGGAGCG 1901 GAAAAGCCCT TGTCCGCATC AGGCAGACGG GAAAAAACAG CGGCACAATC 1951 GACAATACCG GCGGCACACA TACCGCCGAC CTCTCCCGAT TCCCCATCAC 2001 CGCGCGCACA ACGGCAATCA AAGGCAGGTT TGAAGGAAGC CGCTTCCTCC 2051 CCTACCACAC GCGCAACCAA ATCAACGGCG GCGCGCTTGA CGGCAAAGCC 2101 CCGATACTCG GTTACGCCGA AGACCCCGTC GAACTTTTTT TTATGCACAT 2151 CCAAGGCTCG GGCCGTCTGA AAACCCCGTC CGGCAAATAC ATCCGCATCG 2201 GCTATGCCGA CAAAAACGAA CATCCCTACG TTTCCATCGG ACGCTATATG 2251 GCGGACAAAG GCTACCTCAA GCTCGGGCAG ACCTCGATGC AGGGCATCAA 2301 AGCCTATATG CGGCAAAATC CGCAACGCCT CGCCGAAGTT TTGGGTCAAA 2351 ACCCCAGCTA TATCTTTTTC CGCGAGCTTG CCGGAAGCAG CAATGACGGT 2401 CCCGTCGGCG CACTGGGCAC GCCGTTGATG GGGGAATATG CCGGCGCAGT 2451 CGACCGGCAC TACATTACCT TGGGCGCGCC CTTATTTGTC GCCACCGCCC 2501 ATCCGGTTAC CCGCAAAGCC CTCAACCGCC TGATTATGGC GCAGGATACC 2551 GGCAGCGCGA TTAAAGGCGC GGTGCGCGTG GATTATTTTT GGGGATACGG 2601 CGACGAAGCC GGCGAACTTG CCGGCAAACA GAAAACCACG GGTTACGTCT 2651 GGCAGCTCCT ACCCAACGGT ATGAAGCCCG AATACCGCCC GTAAAAGCTT 1 MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM 51 AAVSEENTGN GGAAATDKPR NEDEGAQNDM PQNAADTDSL TPNHTPASNM 101 PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG 151 TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL 201 THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV 251 GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ 301 ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP 351 SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS 401 GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY 451 RPTDAEKGGF GVFAGKKEQD GSGGGGCQSK SIQTFPQPDT SVINGPDRPV 501 GIPDPAGTTV GGGGAVYTVV PHLSLPHWAA QDFAKSLQSF RLGCANLKNR 551 QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV 601 LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNSGTI 651 DNTGGTHTAD LSRFPITART TAIKGRFEGS RFLPYHTRNQ INGGALDGKA 701 PILGYAEDPV ELFFMHIQGS GRLKTPSGKY IRIGYADKNE HPYVSIGRYM 751 ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG 801 PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAHPVTRKA LNRLIMAQDT 851 GSAIKGAVRV DYFWGYGDEA GELAGKQKTT GYVWQLLPNG MKPEYRP* ΔG287NZ-953 (SEQ ID NOS: 96 and 97) 1 ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC 51 CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG 101 CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG 151 GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA 201 CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG 251 CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG 301 CCGGCCGGAA ATATGGAAAA CCAAGGCTCG GATGCCGGGG AATCGGAGCA 351 GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG 401 ACGATCCGTC GGCAGGCGGG GAAAACCACG GCAATACGGC TGCCCAAGGT 451 ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC 501 TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA 551 ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG 601 ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA 651 AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA 701 GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC 751 GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT 801 CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC 851 GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG 901 GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC 951 CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG 1001 CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT 1051 TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT 1101 GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG 1151 CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC 1201 GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG 1251 AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG 1301 GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT 1351 CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA 1401 AGAGCAGGAT GGATCCGGAG GAGGAGGAGC CACCTACAAA GTGGACGAAT 1451 ATCACGCCAA CGCCCGTTTC GCCATCGACC ATTTCAACAC CAGCACCAAC 1501 GTCGGCGGTT TTTACGGTCT GACCGGTTCC GTCGAGTTCG ACCAAGCAAA 1551 ACGCGACGGT AAAATCGACA TCACCATCCC CGTTGCCAAC CTGCAAAGCG 1601 GTTCGCAACA CTTTACCGAC CACCTGAAAT CAGCCGACAT CTTCGATGCC 1651 GCCCAATATC CGGACATCCG CTTTGTTTCC ACCAAATTCA ACTTCAACGG 1701 CAAAAAACTG GTTTCCGTTG ACGGCAACCT GACCATGCAC GGCAAAACCG 1751 CCCCCGTCAA ACTCAAAGCC GAAAAATTCA ACTGCTACCA AAGCCCGATG 1801 GCGAAAACCG AAGTTTGCGG CGGCGACTTC AGCACCACCA TCGACCGCAC 1851 CAAATGGGGC GTGGACTACC TCGTTAACGT TGGTATGACC AAAAGCGTCC 1901 GCATCGACAT CCAAATCGAG GCAGCCAAAC AATAAAAGCT T 1 MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM 51 AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM 101 PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG 151 TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL 201 THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV 251 GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ 301 ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP 351 SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS 401 GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY 451 RPTDAEKGGF GVFAGKKEQD GSGGGGATYK VDEYHANARF AIDHFNTSTN 501 VGGFYGLTGS VEFDQAKRDG KIDITIPVAN LQSGSQHFTD HLKSADIFDA 551 AQYPDIRFVS TKFNFNGKKL VSVDGNLTMH GKTAPVKLKA EKFNCYVSPM 601 AKTEVCGGDF STTIDRTKWG VDYLVNVGMT KSVRIDIQIE AAKQ* ΔG287NZ-961 (SEQ ID NOS: 98 and 99) 1 ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC 51 CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG 101 CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG 151 GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA 201 CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG 251 CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG 301 CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGCCGGGG AATCGGAGCA 351 GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG 401 ACGATCCGTC GGCAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT 451 ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC 501 TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA 551 ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG 601 ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA 651 AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GAAAAATTCA 701 GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC 751 GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT 801 CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC 851 GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG 901 GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC 951 CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG 1001 CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA ACGGCAACCT 1051 TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT 1101 GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG 1151 CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC 1201 GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG 1251 AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG 1301 GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT 1351 CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA 1401 AGAGCAGGAT GGATCCGGAG GAGGAGGAGC CACAAACGAC GACGATGTTA 1451 AAAAAGCTGC CACTGTGGCC ATTGCTGCTG CCTACAACAA TGGCCAAGAA 1501 ATCAACGGTT TCAAAGCTGG AGAGACCATC TACGACATTG ATGAAGACGG 1551 CACAATTACC AATGAAGACG CAACTGCAGC CGATGTTGAA GCCGACGACT 1601 TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA CTAACCTGAC CAAAACCGTC 1651 AATGAAAACA AACAAAACGT CGATGCCAAA GTAAAAGCTG CAGAATCTGA 1701 AATAGAAAAG TTAACAACCA AGTTAGCAGA CACTGATGCC GCTTTAGCAG 1751 ATACTGATGC CGCTCTGGAT GCAACCACCA ACGCCTTGAA TAAATTGGGA 1801 GAAAATATAA CGACATTTGC TGAAGAGACT AAGACAAATA TCGTAAAAAT 1851 TGATGAAAAA TTAGAAGCCG TGGCAAATAC CGTCGACAAG CATGCCGAAG 1901 CATTCAACGA TATCGCCGAT TCATTGGATG AAACCAACAC TAAGGCAGAC 1951 GAAGCCGTCA AAACCGCCAA TGAAGCCAAA CAGACGGCCG AAGAAACCAA 2001 ACAAAACGTC GATGCCAAAG TAAAAGCTGC AGAAACTGCA GCAGGCAAAG 2051 CCGAAGCTGC CGCTGGCACA GCTAATACTG CAGCCGACAA GGCCGAAGCT 2101 GTCGCTGCAA AAGTTACCGA CATCAAAGCT GATATCGCTA CGAACAAAGA 2151 TAATATTGCT AAAAAAGCAA ACAGTGCCGA CGTGTACACC AGAGAAGAGT 2201 CTGACAGCAA ATTTGTCAGA ATTGATGGTC TGAACGCTAC TACCGAAAAA 2251 TTGGACACAC GCTTGGCTTC TGCTGAAAAA TCCATTGCCG ATCACGATAC 2301 TCGCCTGAAC GGTTTGGATA AAACAGTGTC AGACCTGCGC AAAGAAACCC 2351 GCCAAGGCCT TGCTGAAAAA GCCGCGCTCT CCGGTCTGTT CCAACCTTAC 2401 AACGTGGGTC GGTTCAATGT AACGGCTGCA GTCGGCGGCT ACAAATCCGA 2451 ATCGGCAGTC GCCATCGGTA CCGGCTTCCG CTTTACCGAA AACTTTGCCG 2501 CCAAAGCAGG CGTGGCAGTC GGCACTTCGT CCGGTTCTTC CGCAGCCTAC 2551 CATGTCGGCG TCAATTACGA GTGGTAAAAG CTT 1 MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM 51 AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM 101 PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG 151 TNQAENNQTA GSQMPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL 201 THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV 251 GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ 301 ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP 351 SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS 401 GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY 451 RPTDAEKGGF GVFAGKKEQD GSGGGGATND DDVKKAATVA IAAAYNNGQE 501 INGFKAGETI YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV 551 NENKQNVDAK VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG 601 ENITTFAEET KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD 651 EAVKTANEAK QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA 701 VAAKVTDIKA DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK 751 LDTRLASAEK SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY 801 NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY 851 HVGVNYEW* ΔG983 and Hybrids

Bactericidal titres generated in response to ΔG983 (His-fusion) were measured against various strains, including the homologous 2996 strain:

2996 NGH38 BZ133 ΔG983 512 128 128

ΔG983 was also expressed as a hybrid, with ORF46.1 (SEQ ID NOS: 100 and 101), 741 (SEQ ID NOS: 102 and 103), 961 (SEQ ID NOS: 104 and 105) or 961 c(SEQ ID NOS: 106 and 107) at its C-terminus:

ΔG983-OR746.1 1 ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA 51 CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA 101 AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC 151 GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT 201 GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA 251 ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA 301 GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT 351 GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG 401 CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA 451 GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA 501 TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA 551 TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT 601 GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT 651 GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC 701 GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC 751 CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA 801 CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA 851 GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC 901 ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT 951 ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG 1001 GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG 1051 GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT 1101 GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA 1151 ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC 1201 ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT 1251 GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG 1301 ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA 1351 CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC 1401 CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA 1451 TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG 1501 GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA 1551 ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG 1601 CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC 1651 GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT 1701 GGACGCAGCA GGCAAGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG 1751 ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG 1801 GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC 1851 CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG 1901 GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA 1951 GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC 2001 TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GGCGTGGAAC 2051 AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA 2101 TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA 2151 TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG 2201 TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC 2251 GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG 2301 CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC 2351 GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT 2401 GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA 2451 AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA 2501 CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT 2551 GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT 2601 CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG 2651 AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG 2701 GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG 2751 CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA 2801 GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA 2851 CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT 2901 TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA 2951 CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC 3001 AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT 3051 CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC 3101 AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAC 3151 GGTGGCGGAG GCACTGGATC CTCAGATTTG GCAAACGATT CTTTTATCCG 3201 GCAGGTTCTC GACCGTCAGC ATTTCGAACC CGACGGGAAA TACCACCTAT 3251 TCGGCAGCAG GGGGGAACTT GCCGAGCGCA GCGGCCATAT CGGATTGGGA 3301 AAAATACAAA GCCATCAGTT GGGCAACCTG ATGATTCAAC AGGCGGCCAT 3351 TAAAGGAAAT ATCGGCTACA TTGTCCGCTT TTCCGATCAC GGGCACGAAG 3401 TCCATTCCCC CTTCGACAAC CATGCCTCAC ATTCCGATTC TGATGAAGCC 3451 GGTAGTCCCG TTGACGGATT TAGCCTTTAC CGCATCCATT GGGACGGATA 3501 CGAACACCAT CCCGCCGACG GCTATGACGG GCCACAGGGC GGCGGCTATC 3551 CCGCTCCCAA AGGCGCGAGG GATATATACA GCTACGACAT AAAAGGCGTT 3601 GCCCAAAATA TCCGCCTCAA CCTGACCGAC AACCGCAGCA CCGGACAACG 3651 GCTTGCCGAC CGTTTCCACA ATGCCGGTAG TATGCTGACG CAAGGAGTAG 3701 GCGACGGATT CAAACGCGCC ACCCGATACA GCCCCGAGCT GGACAGATCG 3751 GGCAATGCCG CCGAAGCCTT CAACGGCACT GCAGATATCG TTAAAAACAT 3801 CATCGGCGCG GCAGGAGAAA TTGTCGGCGC AGGCGATGCC GTGCAGGGCA 3851 TAAGCGAAGG CTCAAACATT GCTGTCATGC ACGGCTTGGG TCTGCTTTCC 3901 ACCGAAAACA AGATGGCGCG CATCAAOGAT TTGGCAGATA TGGCGCAACT 3951 CAAAGACTAT GCCGCAGCAG CCATCCGCGA TTGGGCAGTC CAAAACCCCA 4001 ATGCCGCACA AGGCATAGAA GCCGTCAGCA ATATCTTTAT GGCAGCCATC 4051 CCCATCAAAG GGATTGGAGC TGTTCGGGGA AAATACGGCT TGGGCGGCAT 4101 CACGGCACAT CCTATCAAGC GGTCGCAGAT GGGCGCGATC GCATTGCCGA 4151 AAGGGAAATC CGCCGTCAGC GACAATTTTG CCGATGCGGC ATACGCCAAA 4201 TACCCGTCCC CTTACCATTC CCGAAATATC CGTTCAAACT TGGAGCAGCG 4251 TTACGGCAAA GAAAACATCA CCTCCTCAAC CGTGCCGCCG TCAAACGGCA 4301 AAAATGTCAA ACTGGCAGAC CAACGCCACC CGAAGACAGG CGTACCGTTT 4351 GACGGTAAAG GGTTTCCGAA TTTTGAGAAG CACGTGAAAT ATGATACGCT 4401 CGAGCACCAC CACCACCACC ACTGA 1 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD 51 VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV 101 GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE 151 ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD 201 ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD 251 LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF 301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP 351 GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG 401 TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG 451 PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT 501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT 551 DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK 601 GAGYLNSTGR RVPFLSAAKI GODYSFFTNI ETDGGLLASL DSVEKTAGSE 651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE 701 SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA 751 ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG 801 VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF 851 AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL 901 GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG 951 LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR 1001 NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD 1051 GGGGTGSSDL ANDSFIRQVL DRQHFEPDGK YHLFGSRGEL AERSGHIGLG 1101 KIQSHQLGNL MIQQAAIKGN IGYIVRFSDH GHEVHSPFDN HASHSDSDEA 1151 GSPVDGFSLY RIHWDGYEHH PADGYDGPQG GGYPAPKGAR DITSYDIKGV 1201 AQNIRLNLTD NRSTGQRLAD RFHNAGSMLT QGVGDGFKRA TRYSPELDRS 1251 GNAAEAFNGT ADIVKNIIGA AGEIVGAGDA VQGISEGSNI AVMHGLGLLS 1301 TENKMARIND LADMAQLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI 1351 PIKGIGAVRG KYGLGGITAH PIKRSQMGAI ALPKGKSAVS DNFADAAYAK 1401 YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGKNVKLAD QRHPKTGVPF 1451 DGKGFPNFEK HVKYDTLEHH HHHH* ΔG983-741 1 ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA 51 CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA 101 AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC 151 GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT 201 GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA 251 ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTACCGGTA 301 GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT 351 GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG 401 CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA 451 GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA 501 TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA 551 TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT 601 GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT 651 GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC 701 GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC 751 CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA 801 CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA 851 GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC 901 ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT 951 ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG 1001 GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG 1051 GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT 1101 GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA 1151 ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC 1201 ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT 1251 GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG 1301 ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA 1351 CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC 1401 CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA 1451 TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG 1501 GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA 1551 ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG 1601 CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC 1651 GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT 1701 GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG 1751 ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG 1801 GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC 1851 CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG 1901 GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA 1951 GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC 2001 TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC 2051 AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA 2101 TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA 2151 TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG 2201 TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC 2251 GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG 2301 CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC 2351 GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT 2401 GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA 2451 AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA 2501 CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT 2551 GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT 2601 CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG 2651 AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCAAACTG 2701 GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG 2751 CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA 2801 GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA 2851 CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT 2901 TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA 2951 CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC 3001 AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT 3051 CCACAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC 3101 AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG 3151 GGATCCGGAG GGGGTGGTGT CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA 3201 TGCACTAACC GCACCGCTCG ACCATAAAGA CAAAGGTTTG CAGTCTTTGA 3251 CGCTGGATCA GTCCGTCAGG AAAAACGAGA AACTGAAGCT GGCGGCACAA 3301 GGTGCGGAAA AAACTTATGG AAACGGTGAC AGCCTCAATA CGGGCAAATT 3351 GAAGAACGAC AAGGTCAGCC GTTTCGACTT TATCCGCCAA ATCGAAGTGG 3401 ACGGGCAGCT CATTACCTTG GAGAGTGGAG AGTTCCAAGT ATACAAACAA 3451 AGCCATTCCG CCTTAACCGC CTTTCAGACC GAGCAAATAC AAGATTCGGA 3501 GCATTCCGGG AAGATGGTTG CGAAACGCCA GTTCAGAATC GGCGACATAG 3551 CGGGCGAACA TACATCTTTT GACAAGCTTC CCGAAGGCGG CAGGGCGACA 3601 TATCGCGGGA CGGCGTTCGG TTCAGACGAT GCCGGCGGAA AACTGACCTA 3651 CACCATAGAT TTCGCCGCCA AGCAGGGAAA CGGCAAAATC GAACATTTGA 3701 AATCGCCAGA ACTCAATGTC GACCTGGCCG CCGCCGATAT CAAGCCGGAT 3751 GGAAAACGCC ATGCCGTCAT CAGCGGTTCC GTCCTTTACA ACCAAGCCGA 3801 GAAAGGCAGT TACTCCCTCG GTATCTTTGG CGGAAAAGCC CAGGAAGTTG 3851 CCGGCAGCGC GGAAGTGAAA ACCGTAAACG GCATACGCCA TATCGGCCTT 3901 GCCGCCAAGC AACTCGAGCA CCACCACCAC CACCACTGA 1 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD 51 VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLRPAIE AGYTGRGVEV 101 GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE 151 ASFDDEAVIR TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD 201 ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD 251 LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF 301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP 351 GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG 401 TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG 451 PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT 501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT 551 DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK 601 GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE 651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE 701 SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA 751 ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG 801 VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF 851 AGIRHDAGDI GYLRGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL 901 GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG 951 LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR 1001 NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE 1051 GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLRLAAQ 1101 GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ 1151 SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT 1201 YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD 1251 GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL 1301 AAKQLEHHHH HH* ΔG983-961 1 ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA 51 CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA 101 AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC 151 GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT 201 GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA 251 ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA 301 GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT 351 GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG 401 CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA 451 GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA 501 TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA 551 TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT 601 GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT 651 GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC 701 GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC 751 CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA 801 CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA 851 GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC 901 ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT 951 ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG 1001 GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG 1051 GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT 1101 GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA 1151 ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC 1201 ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT 1251 GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG 1301 ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA 1351 CCCGCGTCQT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC 1401 CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA 1451 TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG 1501 GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA 1551 ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG 1601 CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC 1651 GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT 1701 GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG 1751 ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG 1801 GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC 1851 CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG 1901 GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA 1951 GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC 2001 TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC 2051 AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA 2101 TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA 2151 TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG 2201 TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC 2251 GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG 2301 CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC 2351 GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT 2401 GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA 2451 AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA 2501 CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT 2551 GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT 2601 CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG 2651 AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG 2701 GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG 2751 CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA 2801 GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGGCGAA 2851 CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT 2901 TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA 2951 CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC 3001 AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT 3051 CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC 3101 AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG 3151 GGTGGCGGAG GCACTGGATC CGCCACAAAC GACGACGATG TTAAAAAAGC 3201 TGCCACTGTG GCCATTGCTG CTGCCTACAA CAATGGCCAA GAAATCAACG 3251 GTTTCAAAGC TGGAGAGACC ATCTACGACA TTGATGAAGA CGGCACAATT 3301 ACCAAAAAAG ACGCAACTGC AGCCGATGTT GAAGCCGACG ACTTTAAAGG 3351 TCTGGGTCTG AAAAAAGTCG TGACTAACCT GACCAAAACC GTCAATGAAA 3401 ACAAACAAAA CGTCGATGCC AAAGTAAAAG CTGCAGAATC TGAAATAGAA 3451 AAGTTAACAA CCAAGTTAGC AGACACTGAT GCCGCTTTAG CAGATACTGA 3501 TGCCGCTCTG GATGCAACCA CCAACGCCTT GAATAAATTG GGAGAAAATA 3551 TAACGACATT TGCTGAAGAG ACTAAGACAA ATATCGTAAA AATTGATGAA 3601 AAATTAGAAG CCGTGGCTGA TACCGTCGAC AAGCATGCCG AAGCATTCAA 3651 CGATATCGCC GATTCATTGG ATGAAACCAA CACTAAGGCA GACGAAGCCG 3701 TCAAAACCGC CAATGAAGCC AAACAGACGG CCGAAGAAAC CAAACAAAAC 3751 GTCGATGCCA AAGTAAAAGC TGCAGAAACT GCAGCAGGCA AAGCCGAAGC 3801 TGCCGCTGGC ACAGCTAATA CTGCAGCCGA CAAGGCCGAA GCTGTCGCTG 3851 CAAAAGTTAC CGACATCAAA GCTGATATCG CTACGAACAA AGATAATATT 3901 GCTAAAAAAG CAAACAGTGC CGACGTGTAC ACCAGAGAAG AGTCTGACAG 3951 CAAATTTGTC AGAATTGATG GTCTGAACGC TACTACCGAA AAATTGGACA 4001 CACGCTTGGC TTCTGCTGAA AAATCCATTG CCGATCACGA TACTCGCCTG 4051 AACGGTTTGG ATAAAACAGT GTCAGACCTG CGCAAAGAAA CCCGCCAAGG 4101 CCTTGCAGAA CAAGCCGCGC TCTCCGGTCT GTTCCAACCT TACAACGTGG 4151 GTCGGTTCAA TGTAACGGCT GCAGTCGGCG GCTACAAATC CGAATCGGCA 4201 GTCGCCATCG GTACCGGCTT CCGCTTTACC GAAAACTTTG CCGCCAAAGC 4251 AGGCGTGGCA GTCGGCACTT CGTCCGGTTC TTCCGCAGCC TACCATGTCG 4301 GCGTCAATTA CGAGTGGCTC GAGCACCACC ACCACCACCA CTGA 1 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD 51 VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV 101 GIVDTGESVC SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE 151 ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD 201 ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD 251 LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF 301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP 351 GTEPLEYGSN HcGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG 401 TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG 451 PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT 501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT 551 DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK 601 GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE 651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE 701 SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA 751 ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG 801 VEGEERGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF 851 AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL 901 GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG 951 LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR 1001 NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE 1051 GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI 1101 TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE 1151 KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE 1201 KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN 1251 VDAKVKAAET AAGEAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI 1301 AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL 1351 NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGRFNVTA AVGGYKSESA 1401 VAIGTGFRFT ENFAAKAGVA VGTSSGSSAA YHVGVNYEWL EHHHHHH* ΔG983-961c 1 ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA 51 CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA 101 AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC 151 GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT 201 GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA 251 ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA 301 GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT 351 GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG 401 CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA 451 GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA 501 TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA 551 TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT 601 GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT 651 GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC 701 GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC 751 CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA 801 CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA 851 GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC 901 ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT 951 ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG 1001 GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG 1051 GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT 1101 GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA 1151 ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC 1201 ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT 1251 GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG 1301 ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA 1351 CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC 1401 CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA 1451 TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG 1501 GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA 1551 ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG 1601 CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC 1651 GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT 1701 GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG 1751 ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG 1801 GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC 1851 CGCCACAAAC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG 1901 GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA 1951 GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC 2001 TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC 2051 AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA 2101 TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA 2151 TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG 2201 TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCGTCAA CAGTCTCGCC 2251 GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG 2301 CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC 2351 GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT 2401 GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA 2451 AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA 2501 CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT 2551 GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT 2601 CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG 2651 AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG 2701 GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG 2751 CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA 2801 GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA 2851 CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT 2901 TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA 2951 CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC 3001 AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT 3051 CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC 3101 AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG 3151 GGTGGCGGAG GCACTGGATC CGCCACAAAC GACGACGATG TTAAAAAAGC 3201 TGCCACTGTG GCCATTGCTG CTGCCTACAA CAATGGCCAA GAAATCAACG 3251 GTTTCAAAGC TGGAGAGACC ATCTACGACA TTGATGAAGA CGGCACAATT 3301 ACCAAAAAAG ACGCAACTGC AGCCGATGTT GAAGCCGACG ACTTTAAAGG 3351 TCTGGGTCTG AAAAAAGTCG TGACTAACCT GACCAAAACC GTCAATGAAA 3401 ACAAACAAAA CGTCGATGCC AAAGTAAAAG CTGCAGAATC TGAAATAGAA 3451 AAGTTAACAA CCAAGTTAGC AGACACTGAT GCCGCTTTAG CAGATACTGA 3501 TGCCGCTCTG GATGCAACCA CCAACGCCTT GAATAAATTG GGAGAAAATA 3551 TAACGACATT TGCTGAAGAG ACTAAGACAA ATATCGTAAA AATTGATGAA 3601 AAATTAGAAG CCGTGGCTGA TACCGTCGAC AAGCATGCCG AAGCATTCAA 3651 CGATATCGCC GATTCATTGG ATGAAACCAA CACTAAGGCA GACGAAGCCG 3701 TCAAAACCGC CAATGAAGCC AAACAGACGG CCGAAGAAAC CAAACAAAAC 3751 GTCGATGCCA AAGTAAAAGC TGCAGAAACT GCAGCAGGCA AAGCCGAAGC 3801 TGCCGCTGGC ACAGCTAATA CTGCAGCCGA CAAGGCCGAA GCTGTCGCTG 3851 CAAAAGTTAC CGACATCAAA GCTGATATCG CTACGAACAA AGATAATATT 3901 GCTAAAAAAG CAAACAGTGC CGACGTGTAC ACCAGAGAAG AGTCTGACAG 3951 CAAATTTGTC AGAATTGATG GTCTGAACGC TACTACCGAA AAATTGGACA 4001 CACGCTTGGC TTCTGCTGAA AAATCCATTG CCGATCACGA TACTCGCCTG 4051 AACGGTTTGG ATAAAACAGT GTCAGACCTG CGCAAAGAAA CCCGCCAAGG 4101 CCTTGCAGAA CAAGCCGCGC TCTCCGGTCT GTTCCAACCT TACAACGTGG 4151 GTCTCGAGCA CCACCACCAC CACCACTGA 1 MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD 51 VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLEPAIE AGYTGRGVEV 101 GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE 151 ASFDDEAVIE TEAKPTDIRH VKSIGHIDLV SHIIGGRSVD GRPAGGIAPD 201 ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD 251 LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF 301 IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP 351 GTEPLEYGSN HCGITAMNCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG 401 TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG 451 PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT 501 GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT 551 DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK 601 GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE 651 GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE 701 SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA 751 ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG 801 VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF 851 AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL 901 GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG 951 LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR 1001 NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE 1051 GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI 1101 TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE 1151 KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE 1201 KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVETANEA KQTAEETKQN 1251 VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI 1301 AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL 1351 NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGLEHHHH HH* ΔG741 and Hybrids

Bactericidal titres generated in response to ΔG741 (His-fusion) were measured against various strains, including the homologous 2996 strain:

2996 MC58 NGH38 F6124 BZ133 ΔG741 512 131072 >2048 16384 >2048

As can be seen, the ΔG741-induced anti-bactericidal titre is particularly high against heterologous strain MC58.

ΔG741 was also fused directly in-frame upstream of proteins 961 (SEQ ID NOS:108 and 109), 961c, (SEQ ID NOS:110 and 111), 983 (SEQ ID NOS:112 and 113) and ORF46.1 (SEQ ID NOS:114 and 115):

ΔG741-961 1 ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC 51 GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG 101 TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT 151 TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT 201 CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA 251 CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA 301 ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT 351 GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT 401 CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG 451 TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC 501 CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA 551 ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC 601 GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC 651 CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG 701 TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC 751 GAGGGTGGCG GAGGCACTGG ATCCGCCACA AACGACGACG ATGTTAAAAA 801 AGCTGCCACT GTGGCCATTG CTGCTGCCTA CAACAATGGC CAAGAAATCA 851 ACGGTTTCAA AGCTGGAGAG ACCATCTACG ACATTGATGA AGACGGCACA 901 ATTACCAAAA AAGACGCAAC TGCAGCCGAT GTTGAAGCCG ACGACTTTAA 951 AGGTCTGGGT CTGAAAAAAG TCGTGACTAA CCTGACCAAA ACCGTCAATG 1001 AAAACAAACA AAACGTCGAT GCCAAAGTAA AAGCTGCAGA ATCTGAAATA 1051 GAAAAGTTAA CAACCAAGTT AGCAGACACT GATGCCGCTT TAGCAGATAC 1101 TGATGCCGCT CTGGATGCAA CCACCAACGC CTTGAATAAA TTGGGAGAAA 1151 ATATAACGAC ATTTGCTGAA GAGACTAAGA CAAATATCGT AAAAATTGAT 1201 GAAAAATTAG AAGCCGTGGC TGATACCGTC GACAAGCATG CCGAAGCATT 1251 CAACGATATC GCCGATTCAT TGGATGAAAC CAACACTAAG GCAGACGAAG 1301 CCGTCAAAAC CGCCAATGAA GCCAAACAGA CGGCCGAAGA AACCAAACAA 1351 AACGTCGATG CCAAAGTAAA AGCTGCAGAA ACTGCAGCAG GCAAAGCCGA 1401 AGCTGCCGCT GGCACAGCTA ATACTGCAGC CGACAAGGCC GAAGCTGTCG 1451 CTGCAAAAGT TACCGACATC AAAGCTGATA TCGCTACGAA CAAAGATAAT 1501 ATTGCTAAAA AAGCAAACAG TGCCGACGTG TACACCAGAG AAGAGTCTGA 1551 CAGCAAATTT GTCAGAATTG ATGGTCTGAA CGCTACTACC GAAAAATTGG 1601 ACACACGCTT GGCTTCTGCT GAAAAATCCA TTGCCGATCA CGATACTCGC 1651 CTGAACGGTT TGGATAAAAC AGTGTCAGAC CTGCGCAAAG AAACCCGCCA 1701 AGGCCTTGCA GAACAAGCCG CGCTCTCCGG TCTGTTCCAA CCTTACAACG 1751 TGGGTCGGTT CAATGTAACG GCTGCAGTCG GCGGCTACAA ATCCGAATCG 1801 GCAGTCGCCA TCGGTACCGG CTTCCGCTTT ACCGAAAACT TTGCCGCCAA 1851 AGCAGGCGTG GCAGTCGGCA CTTCGTCCGG TTCTTCCGCA GCCTACCATG 1901 TCGGCGTCAA TTACGAGTGG CTCGAGCACC ACCACCACCA CCACTGA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL 101 TAFQTEQIQD SEHSGKHVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA 151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA 201 VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL 251 EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT 301 ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI 351 EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID 401 EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ 451 NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN 501 IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR 551 LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGRFNVT AAVGGYKSES 601 AVAIGTGFRF TENFAAKAGV AVGTSSGSSA AYHVGVNYEW LEEHHHHH* ΔG741-961c 1 ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC 51 GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG 101 TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT 151 TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT 201 CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA 251 CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA 301 ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT 351 GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT 401 CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG 451 TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC 501 CGCCAAGCAG GGAAACGGCA AAAACAAACA TTTGAAATCG CCAGAACTCA 551 ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC 601 GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC 651 CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG 701 TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC 751 GAGGGTGGCG GAGGCACTGG ATCCGCCACA AACGACGACG ATGTTAAAAA 801 AGCTGCCACT GTGGCCATTG CTGCTGCCTA CAACAATGGC CAAGAAATCA 851 ACGGTTTCAA AGCTGGAGAG ACCATCTACG ACATTGATGA AGACGGCACA 901 ATTACCAAAA AAGACGCAAC TGCAGCCGAT GTTGAAGCCG ACGACTTTAA 951 AGGTCTGGGT CTGAAAAAAG TCGTGACTAA CCTGACCAAA ACCGTCAATG 1001 AAAACAAACA AAACGTCGAT GCCAAAGTAA AAGCTGCAGA ATCTGAAATA 1051 GAAAAGTTAA CAACCAAGTT AGCAGACACT GATGCCGCTT TAGCAGATAC 1101 TGATGCCGCT CTGGATGCAA CCACCAACGC CTTGAATAAA TTGGGAGAAA 1151 ATATAACGAC ATTTGCTGAA GAGACTAAGA CAAATATCGT AAAAATTGAT 1201 GAAAAATTAG AAGCCGTGGC TGATACCGTC GACAAGCATG CCGAAGCATT 1251 CAACGATATC GCCGATTCAT TGGATGAAAC CAACACTAAG GCAGACGAAG 1301 CCGTCAAAAC CGCCAATGAA GCCAAATCGA CGGCCGAAGA AACCAAACAA 1351 AACGTCGATG CCAAAGTAAA AGCTGCAGAA ACTGCAGCAG GCAAAGCCGA 1401 AGCTGCCGCT GGCACAGCTA ATACTGCAGC CGACAAGGCC GAAGCTGTCG 1451 CTGCAAAAGT TACCGACATC AAAGCTGATA TCGCTACGAA CAAAGATAAT 1501 ATTGCTAAAA AAGCAAACAG TGCCGACGTG TACACCAGAG AAGAGTCTGA 1551 CAGCAAATTT GTCAGAATTG ATGGTCTGAA CGCTACTACC GAAAAATTGG 1601 ACACACGCTT GGCTTCTGCT GAAAAATCCA TTGCCGATCA CGATACTCGC 1651 CTGAACGGTT TGGATAAAAC AGTGTCAGAC CTGCGCAAAG AAACCCGCCA 1701 AGGCCTTGCA GAACAAGCCG CGCTCTCCGG TCTGTTCCAA CCTTACAACG 1751 TGGGTCTCGA GCACCACCAC CACCACCACT GA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL 101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA 151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA 201 VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL 251 EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT 301 ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI 351 EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID 401 EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ 451 NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN 501 IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR 551 LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGLEHHH HHH* ΔG741-983 1 ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC 51 GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG 101 TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT 151 TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT 201 CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA 251 CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA 301 ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT 351 GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT 401 CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG 451 TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC 501 CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CAAGAAATCA 551 ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC 601 GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC 651 CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGANG 701 TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC 751 GAGGGATCCG GCGGAGGCGG CACTTCTGCG CCCGACTTCA ATGCAGGCGG 801 TACCGGTATC GGCAGCAACA GCAGAGCAAC AACAGCGAAA TCAGCAGCAG 851 TATCTTACGC CGGTATCAAG AACGAAATGT GCAAAGACAG AAGCATGCTC 901 TGTGCCGGTC GGGATGACGT TGCGGTTACA GACAGGGATG CCAAAATCAA 951 TGCCCCCCCC CCGAATCTGC ATACCGGAGA CTTTCCAAAC CCAAATGACG 1001 CATACAAGAA TTTGATCAAC CTCAAACCTG CAATTGAAGC AGGCTATACA 1051 GGACGCGGGG TAGAGGTAGG TATCGTCGAC ACAGGCGAAT CCGTCGGCAG 1101 CATATCCTTT CCCGAACTGT ATGGCAGAAA AGAACACGGC TATAACGAAA 1151 ATTACCAAAA CTATACGGCG TATATGCGGA AGGAAGCGCC TGAAGACGGA 1201 GGCGGTAAAG ACATTGAAGC TTCTTTCGAC GATGAGGCCG TTATAGAGAC 1251 TGAAGCAAAG CCGACGGATA TCCGCCACGT AAAAGAAATC GGACACATCG 1301 ATTTGGTCTC CCATATTATT GGCGGGCGTT CCGTGGACGG CAGACCTGCA 1351 GGCGGTATTG CGCCCGATGC GACGCTACAC ATAATGAATA CGAATGATGA 1401 AACCAAGAAC GAAATGATGG TTGCAGCCAT CCGCAATGCA TGGGTCAAGC 1451 TGGGCGAACG TGGCGTGCGC ATCGTCAATA ACAGTTTTGG AACAACATCG 1501 AGGGCAGGCA CTGCCGACCT TTTCCAAATA GCCAATTCGG AGGAGCAGTA 1551 CCGCCAAGCG TTGCTCGACT ATTCCGGCGG TGATAAAACA GACGAGGGTA 1601 TCCGCCTGAT GCAACAGAGC GATTACGGCA ACCTGTCCTA CCACATCCGT 1651 AATAAAAACA TGCTTTTCAT CTTTTCGACA GGCAATGACG CACAAGCTCA 1701 GCCCAACACA TATGCCCTAT TGCCATTTTA TGAAAAAGAC GCTCAAAAAG 1751 GCATTATCAC AGTCGCAGGC GTAGACCGCA GTGGAGAAAA GTTCAAACGG 1801 GAAATGTATG GAGAACCGGG TACAGAACCG CTTGAGTATG GCTCCAACCA 1851 TTGCGGAATT ACTGCCATGT GGTGCCTGTC GGCACCCTAT GAAGCAAGCG 1901 TCCGTTTCAC CCGTACAAAC CCGATTCAAA TTGCCGGAAC ATCCTTTTCC 1951 GCACCCATCG TAACCGGCAC GGCGGCTCTG CTGCTGCAGA AATACCCGTG 2001 GATGAGCAAC GACAACCTGC GTACCACGTT GCTGACGACG GCTCAGGACA 2051 TCGGTGCAGT CGGCGTGGAC AGCAAGTTCG GCTGGGGACT GCTGGATGCG 2101 GGTAAGGCCA TGAACGGACC CGCGTCCTTT CCGTTCGGCG ACTTTACCGC 2151 CGATACGAAA GGTACATCCG ATATTGCCTA CTCCTTCCGT AACGACATTT 2201 CAGGCACGGG CGGCCTGATC AAAAAAGGCG GCAGCCAACT GCAACTGCAC 2251 GGCAACAACA CCTATACGGG CAAAACCATT ATCGAAGGCG GTTCGCTGGT 2301 GTTGTACGGC AACAACAAAT CGGATATGCG CGTCGAAACC AAAGGTGCGC 2351 TGATTTATAA CGGGGCGGCA TCCGGCGGCA GCCTGAACAG CGACGGCATT 2401 GTCTATCTGG CAGATACCGA CCAATCCGGC GCAAACGAAA CCGTACACAT 2451 CAAAGGCAGT CTGCAGCTGG ACGGCAAAGG TACGCTGTAC ACACGTTTGG 2501 GCAAACTGCT GAAAGTGGAC GGTACGGCGA TTATCGGCGG CAAGCTGTAC 2551 ATGTCGGCAC GCGGCAAGGG GGCAGGCTAT CTCAACAGTA CCGGACGACG 2601 TGTTCCCTTC CTGAGTGCCG CCAAAATCGG GCAGGATTAT TCTTTCTTCA 2651 CAAACATCGA AACCGACGGC GGCCTGCTGG CTTCCCTCGA CAGCGTCGAA 2701 AAAACAGCGG GCAGTGAAGG CGACACGCTG TCCTATTATG TCCGTCGCGG 2751 CAATGCGGCA CGGACTGCTT CGGCAGCGGC ACATTCCGCG CCCGCCGGTC 2801 TGAAACACGC CGTAGAACAG GGcGGCAGCA ATCTGGAAAA CCTGATGGTC 2851 GAACTGGATG CCTCCGAATC ATCCGCAACA CCCGAGACGG TTGAAACTGC 2901 GGCAGCCGAC CGCACAGATA TGCCGGGCAT CCGCCCCTAC GGCGCAACTT 2951 TCCGCGCAGC GGCAGCCGTA CAGCATGCGA ATGCCGCCGA CGGTGTACGC 3001 ATCTTCAACA GTCTCGCCGC TACCGTCTAT GCCGACAGTA CCGCCGCCCA 3051 TGCCGATATG CAGGGACGCC GCCTGAAAGC CGTATCGGAC GGGTTGGACC 3101 ACAACGGCAC GGGTCTGCGC GTCATCGCGC AAACCCAACA GGACGGTGGA 3151 ACGTGGGAAC AGGGCGGTGT TGAAGGCAAA ATGCGCGGCA GTACCCAAAC 3201 CGTCGGCATT GCCGCGAAAA CCGGCGAAAA TACGACAGCA GCCGCCACAC 3251 TGGGCATGGG ACGCAGCACA TGGAGCGAAA ACAGTGCAAA TGCAAAAACC 3301 GACAGCATTA GTCTGTTTGC AGGCATACGG CACGATGCGG GCGATATCGG 3351 CTATCTCAAA GGCCTGTTCT CCTACGGACG CTACAAAAAC AGCATCAGCC 3401 GCAGCACCGG TGCGGACGAA CATGCGGAAG GCAGCGTCAA CGGCACGCTG 3451 ATGCAGCTGG GCGCACTGGG CGGTGTCAAC GTTCCGTTTG CCGCAACGGG 3501 AGATTTGACG GTCGAAGGCG GTCTGCGCTA CGACCTGCTC AAACAGGATG 3551 CATTCGCCGA AAAAGGCAGT GCTTTGGGCT GGAGCGGcAA CAGCCTCACT 3601 GAAGGCACGC TGGTCGGACT CGCGGGTCTG AAGCTGTCGC AACCCTTGAG 3651 CGATAAAGCC GTCCTGTTTG CAACGGCGGG CGTGGAAcGC GACCTGAACG 3701 GACGCGACTA CACGGTAACG GGCGGCTTTA CCGGCGCGAC TGCAGCAACC 3751 GGCAAGACGG GGGCACGCAA TATGCCGCAC ACCCGTCTGG TTGCCGGCCT 3801 GGGCGCGGAT GTCGAATTCG GCAACGGCTG GAACGGCTTG GCACGTTACA 3851 GCTACGCCGG TTCCAAACAG TACGGCAACC ACAGCGGACG AGTCGGCGTA 3901 GGCTACCGGT TCCTCGAGCA CCACCACCAC CACCACTGA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL 101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA 151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA 201 VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL 251 EGSGGGGTSA PDFNAGGTGI GSNSRATTAK SAAVSYAGIK NEMCKDRSML 301 CAGRDDVAVT DRDAKINAPP PNLHTGDFPN PNDAYKNLIN LKPAIEAGYT 351 GRGVEVGIVD TGESVGSISF PELYGRKEHG YNENYKNYTA YMRKEAPEDG 401 GGKDIEASFD DEAVIETEAK PTDIRHVKEI GHIDLVSHII GGRSVDGRPA 451 GGIAPDATLH IMNTNDETKN EMMVAAIRNA WVKLGERGVR IVNNSFGTTS 501 RAGTADLFQI ANSEEQYRQA LLDYSGGDKT DEGIRLMQQS DYGNLSYHIR 551 NKNMLFIFST GNDAQAQPNT YALLPFYEKD AQKGIITVAG VDRSGEKFKR 601 EMYGEPGTEP LEYGSNHCGI TAMWCLSAPY EASVRFTRTN PIQIAGTSFS 651 APIVTGTAAL LLQKYPWMSN DNLRTTLLTT AQDIGAVGVD SKFGWGLLDA 701 GKAMNGPASF PFGDFTADTK GTSDIAYSFR NDISGTGGLI KKGGSQLQLH 751 GNNTYTGKTI IEGGSLVLYG NNKSDMRVET KGALIYNGAA SGGSLNSDGI 801 VYLADTDQSG ANETVHIKGS LQLDGKGTLY TRLGKLLKVD GTAIIGGKLY 851 MSARGKGAGY LNSTGRRVPF LSAAKIGQDY SFFTNIETDG GLLASLDSVE 901 KTAGSEGDTL SYYVRRGNAA RTASAAAHSA PAGLKHAVEQ GGSNLENLMV 951 ELDASESSAT PETVETAAAD RTDMPGIRPY GATFRAAAAV QHANAADGVR 1001 IFNSLAATVY ADSTAAHADM QGRRLKAVSD GLDHNGTGLR VIAQTQQDGG 1051 TWEQGGVEGK MRGSTQTVGI AAKTGENTTA AATLGMGRST WSENSANAKT 1101 DSISLFAGIR HDAGDIGYLK GLFSYGRYKN SISRSTGADE HAEGSVNGTL 1151 MQLGALGGVN VPFAATGDLT VEGGLRYDLL KQDAFAEKGS ALGWSGNSLT 1201 EGTLVGLAGL KLSQPLSDKA VLFATAGVER DLNGRDYTVT GGFTGATAAT 1251 GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSKQ YGNHSGRVGV 1301 GYRFLEHHHH HH* ΔG741-ORF46.1 1 ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC 51 GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG 101 TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT 151 TATGGAAACG GTGACAGCCT CAATACGGGC AAATCGAACA ACGACAAGGT 201 CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA 251 CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA 301 ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT 351 GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT 401 CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG 451 TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC 501 CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA 551 ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC 601 GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC 651 CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG 701 TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC 751 GACGGTGGCG GAGGCACTGG ATCCTCAGAT TTGGCAAACG ATTCTTTTAT 801 CCGGCAGGTT CTCGACCGTC AGCATTTCGA ACCCGACGGG AAATACCACC 851 TATTCGGCAG CAGGGGGGAA CTTGCCGAGC GCAGCGGCCA TATCGGATTG 901 GGAAAAATAC AAAGCCATCA GTTGGGCAAC CTGATGATTC AACAGGCGGC 951 CATTAAAGGA AATATCGGCT ACATTGTCCG CTTTTCCGAT CACGGGCACG 1001 AAGTCCATTC CCCCTTCGAC AACCATGCCT CACATTCCGA TTCTGATGAA 1051 GCCGGTAGTC CCGTTGACGG ATTTAGCCTT TACCGCATCC ATTGGGACGG 1101 ATACGAACAC CATCCCGCCG ACGGCTATGA CGGGCCACAG GGCGGCGGCT 1151 ATCCCGCTCC CAAAGGCGCG AGGGATATAT ACAGCTACGA CATAAAAGGC 1201 GTTGCCCAAA ATATCCGCCT CAACCTGACC GACAACCGCA GCACCGGACA 1251 ACGGCTTGCC GACCGTTTCC ACAATGCCGG TAGTATGCTG ACGCAAGGAG 1301 TAGGCGACGG ATTCAAACGC GCCACCCGAT ACAGCCCCGA GCTGGACAGA 1351 TCGGGCAATG CCGCCGAAGC CTTCAACGGC ACTGCAGATA TCGTTAAAAA 1401 CATCATCGGC GCGGCAGGAG AAATTGTCGG CGCAGGCGAT GCCGTGCAGG 1451 GCATAAGCGA AGGCTCAAAC ATTGCTGTCA TGCACGGCTT GGGTCTGCTT 1501 TCCACCGAAA ACAAGATGGC GCGCATCAAC GATTTGGCAG ATATGGCGCA 1551 ACTCAAAGAC TATGCCGCAG CAGCCATCCG CGATTGGGCA GTCCAAAACC 1601 CCAATGCCGC ACAAGGCATA GAAGCCGTCA GCAATATCTT TATGGCAGCC 1651 ATCCCCATCA AAGGGATTGG AGCTGTTCGG GGAAAATACG GCTTGGGCGG 1701 CATCACGGCA CATCCTATCA AGCGGTCGCA GATGGGCGCG ATCGCATTGC 1751 CGAAAGGGAA ATCCGCCGTC AGCGACAATT TTGCCGATGC GGCATACGCC 1801 AAATACCCGT CCCCTTACCA TTCCCGAAAT ATCCGTTCAA ACTTGGAGCA 1851 GCGTTACGGC AAAGAAAACA TCACCTCCTC AACCGTGCCG CCGTCAAACG 1901 GCAAAAATGT CAAACTGGCA GACCAACGCC ACCCGAAGAC AGGCGTACCG 1951 TTTGACGGTA AAGGGTTTCC GAATTTTGAG AAGCACGTGA AATATGATAC 2001 GCTCGAGCAC CACCACCACC ACCACTGA 1 MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT 51 YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL 101 TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRARYAGTA 151 FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA 201 VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL 251 DGGGGTGSSD LANDSFIRQV LDRQHFEPDG KYHLFGSRGE LAERSGHIGL 301 GKIQSHQLGN LMIQQAAIKG NIGYIVRFSD HGHEVHSPFD NHASHSDSDE 351 AGSPVDGFSL YRIHWDGYEH HPADGYDGPQ GGGYPAPKGA RDIYSYDIKG 401 VAQNIRLNLT DNRSTGQRLA DRFHNAGSML TQGVGDGFKR ATRYSPELDR 451 SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN IAVMHGLGLL 501 STENKMARIN DLADMAQLKD YAAAAIRDWA VQNPNAAQGI EAVSNIFMAA 551 IPIKGIGAVR GKYGLGGITA HPIKRSQMGA IALPKGKSAV SDNFADANYA 601 KYPSPYHSRN IRSNLEQRYG KENITSSTVP PSNGXNVKLA DQRHPKTGVP 651 FDGKGFPNFE KHVKYDTLEH HHHHH*

Example 16 C-Terminal Fusions (‘Hybrids’) with 287/ΔG287

According to the invention, hybrids of two proteins A & B may be either NH₂-A-B—COOH or NH₂—B-A-COOH. The effect of this difference was investigated using protein 287 either C-terminal (in ‘287-His’ form) or N-terminal (in ΔG287 form—sequences shown above) to 919, 953 and ORF46.1. A panel of strains was used, including homologous strain 2996. FCA was used as adjuvant:

287 & 919 287 & 953 287 & ORF46.1 Strain ΔG287-919 919-287 ΔG287-953 953-287 ΔG287-46.1 46.1-287 2996 128000 16000 65536 8192 16384 8192 BZ232 256 128 128 <4 <4 <4 1000 2048 <4 <4 <4 <4 <4 MC58 8192 1024 16384 1024 512 128 NGH38 32000 2048 >2048 4096 16384 4096 394/98 4096 32 256 128 128 16 MenA (F6124) 32000 2048 >2048 32 8192 1024 ManC (BZ133) 64000 >8192 >8192 <16 8192 2048

Better bactericidal titres are generally seen with 287 at the N-terminus (in the ΔG form)

When fused to protein 961 [NH₂-ΔG287-961-COOH—sequence shown above], the resulting protein is insoluble and must be denatured and renatured for purification. Following renaturation, around 50% of the protein was found to remain insoluble. The soluble and insoluble proteins were compared, and much better bactericidal titres were obtained with the soluble protein (FCA as adjuvant):

2996 BZ232 MC58 NGH38 F6I24 BZ133 Soluble 65536 128 4096 >2048 >2048 4096 Insoluble 8192 <4 <4 16 n.d. n.d.

Titres with the insoluble form were, however, improved by using alum adjuvant instead:

Insoluble 32768 128 4096 >2048 >2048 2048

Example 17 N-Terminal Fusions (‘Hybrids’) to 287

Expression of protein 287 as full-length with a C-terminal His-tag, or without its leader peptide but with a C-terminal His-tag, gives fairly low expression levels. Better expression is achieved using a N-terminal GST-fusion.

As an alternative to using GST as an N-terminal fusion partner, 287 was placed at the C-terminus of protein 919 (‘919-287’), of protein 953 (‘953-287’), and of proteins ORF46.1 (‘ORF46.1-287’). In both cases, the leader peptides were deleted, and the hybrids were direct in-frame fusions.

To generate the 953-287 hybrid, the leader peptides of the two proteins were omitted by designing the forward primer downstream from the leader of each sequence; the stop codon sequence was omitted in the 953 reverse primer but included in the 287 reverse primer. For the 953 gene, the 5′ and the 3′ primers used for amplification included a NdeI and a BamHI restriction sites respectively, whereas for the amplification of the 287 gene the 5′ and the 3′ primers included a BamHI and a XhoI restriction sites respectively. In this way a sequential directional cloning of the two genes in pET21b+, using NdeI-BamHI (to clone the first gene) and subsequently BamHI-XhoI (to clone the second gene) could be achieved.

The 919-287 hybrid was obtained by cloning the sequence coding for the mature portion of 287 into the XhoI site at the 3′-end of the 919-His clone in pET21b+. The primers used for amplification of the 287 gene were designed for introducing a SalI restriction site at the 5′- and a XhoI site at the 3′- of the PCR fragment. Since the cohesive ends produced by the SalI and XhoI restriction enzymes are compatible, the 287 PCR product digested with SalI-XhoI could be inserted in the pET21b-919 clone cleaved with XhoI.

The ORF46.1-287 hybrid was obtained similarly.

The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens:

Mixture with 287 Hybrid with 287 919 32000 16000 953 8192 8192 ORF46.1 128 8192

Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained for 919-287 and 953-287:

919 953 ORF46.1 Strain Mixture Hybrid Mixture Hybrid Mixture Hybrid MC58 512 1024 512 1024 — 1024 NGH38 1024 2048 2048 4096 — 4096 BZ232 512 128 1024 16 — — MenA (F6124) 512 2048 2048 32 — 1024 MenC (C11) >2048 n.d. >2048 n.d. — n.d. MenC (BZ133) >4096 >8192 >4096 <16 — 2048

Hybrids of ORF46.1 and 919 were also constructed. Best results (four-fold higher titre) were achieved with 919 at the N-terminus.

Hybrids 919-519His, ORF97-225His and 225-ORF97His were also tested. These gave moderate ELISA fitres and bactericidal antibody responses.

Example 18 The Leader Peptide from ORF4

As shown above, the leader peptide of ORF4 can be fused to the mature sequence of other proteins (e.g. proteins 287 and 919). It is able to direct lipidation in E. coli.

Example 19 Domains in 564

The protein ‘564’ is very large (2073aa), and it is difficult to clone and express it in complete form. To facilitate expression, the protein has been divided into four domains, as shown in FIG. 8 (according to the MC58 sequence):

Domain A B C D Amino Acids 79-360 361-731 732-2044 2045-2073

These domains show the following homologies:

-   -   Domain A shows homology to other bacterial toxins:

gb|AAG03431.1|AE004443_9 probable hemagglutinin [Pseudomonas aeruginosa] (38%) gb|AAC31981.1|(139897) HecA [Pectobacterium chrysanthemi] (45%) emb|CAA36409.1|(X52156) filamentous hemagglutinin [Bordetella pertussis] (31%) gb|AAC79757.1|(AF057695) large supernatant protein1 [Haemophilus ducreyi] (26%) gb|AAA25657.1|(M30186) HpmA precursor [Proteus mirabilis] (29%)

-   -   Domain B shows no homology, and is specific to 564.     -   Domain C shows homology to:

gb|AAF84995.1|AE004032 HA-like secreted protein [Xylella fastidiosa] (33%) gb|AAG05850.1|AE004673 hypothetical protein [Pseudomonas aeruginosa] (27%) gb|AAF68414.1AF237928 putative FHA [Pasteurella multocisida] (23%) gb|AAC79757.1|(AF057695) large supernatant protein1 [Haemophilus ducreyi] (23%) pir||S21010 FHA B precursor [Bordetella pertussis] (20%)

-   -   Domain D shows homology to other bacterial toxins:

gb|AAF84995.1|AE004032_14 HA-like secreted protein [Xylella fastidiosa] (29%)

Using the MC58 strain sequence, good intracellular expression of 564ab was obtained in the form of GST-fusions (no purification) and his-tagged protein; this domain-pair was also expressed as a lipoprotein, which showed moderate expression in the outer membrane/supernatant fraction.

The b domain showed moderate intracellular expression when expressed as a his-tagged product (no purification), and good expression as a GST-fusion.

The c domain showed good intracellular expression as a GST-fusion, but was insoluble. The d domain showed moderate intracellular expression as a his-tagged product (no purification). The cd protein domain-pair showed moderate intracellular expression (no purification) as a GST-fusion.

Good bactericidal assay titres were observed using the c domain and the be pair.

Example 20 The 919 Leader Peptide

The 20mer leader peptide (SEQ ID NO:633) from 919 is discussed in example 1 above:

-   -   MKKYLFRAAL YGIAAAILAA

As shown in example 1, deletion of this leader improves heterologous expression, as does substitution with the ORF4 leader peptide. The influence of the 919 leader on expression was investigated by fusing the coding sequence to the PhoC reporter gene from Morganella morganii [Thaller et al. (1994) Microbiology 140:1341-1350]. The construct (SEQ ID NO:116)was cloned in the pET21-b plasmid between the NdeI and XhoI sites (FIG. 9):

1 MKKYLFRAAL YGIAAAILAA AIPAGNDATT KPDLYYLKNE QAIDSLKLLP 51 PPPEVGSIQF LNDQAMYEKG RMLRNTERGK QAQADADLAA GGVATAFSGA 101 FGYPITEKDS PELYKLLTNM IEDAGDLATR SAKEHYNRIR PFAFYGTETC 151 NTKDQKKLST NGSYPSGHTS IGWATALVLA EVNPANQDAI LERGYQLGQS 201 RVICGYHWQS DVDAARIVGS AAVATLHSDP AFQAQLAKAK QEFAQKSQK*

The level of expression of PhoC from this plasmid is >200-fold lower than that found for the same construct but containing the native PhoC signal peptide. The same result was obtained even after substitution of the T7 promoter with the E. coli Plac promoter. This means that the influence of the 919 leader sequence on expression does not depend on the promoter used.

In order to investigate if the results observed were due to some peculiarity of the 919 signal peptide nucleotide sequence (secondary structure formation, sensitivity to RNAases, etc.) or to protein instability induced by the presence of this signal peptide, a number of mutants were generated. The approach used was a substitution of nucleotides of the 919 signal peptide sequence by cloning synthetic linkers containing degenerate codons. In this way, mutants were obtained with nucleotide and/or amino acid substitutions.

Two different linkers were used, designed to produce mutations in two different regions of the 919 signal peptide sequence, in the first 19 base pairs (L1) (SEQ ID NO:117)and between bases 20-36 (S1) (SEQ ID NO:118).

-   -   L1: 5′ T ATG AAa/g TAc/t c/tTN TTt/c a/cGC GCC GCC CTG TAC GGC         ATC GCC GCC GCC ATC CTC GCC GCC GCG ATC CC 3′     -   S1: 5′ T ATG AAA AAA TAC CTA TTC CGa/g GCN GCN c/tTa/g TAc/t         GGc/g ATC GCC GCC GCC ATC CTC GCC GCC GCG ATC CC 3′

The alignment of some of the mutants obtained is given below.

L1 Mutants:

(SEQ ID NO: 119) 9L1-a ATGAAGAAGTACCTTTTCAGCGCCGCC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (SEQ ID NO: 120) 9L1-e ATGAAAAAATACTTTTTCCGCGCCGCC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (SEQ ID NO: 121) 9L1-d ADGAAAAAATACTTTTTCCGCGCCGCC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (SEQ ID NO: 122) 9L1-f ATGAAAAAATATCTCTTTAGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 123) 919sp ATGAAAAAATACCTATTCCGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 124) 9L1a MKKYLFSAA~~~~~~~~~~~ (SEQ ID NO: 125) 9L1e MKKYFFRAA~~~~~~~~~~~ (SEQ ID NO: 126) 9L1d MKKYFFRAA~~~~~~~~~~~ (SEQ ID NO: 127) 9L1f MKKYLFSAALYGIAAAILAA (SEQ ID NO: 128) 919sp MKKYLFRAALYGIAAAILAA (i.e. native signal peptide)

S1 Mutants:

(SEQ ID NO: 129) 9S1-e ATGAAAAAATACCTATTC..................ATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 130) 9S1-c ATGAAAAAATACCTATTCCGAGCTGCCCAATACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 131) 9S1-b ATGAAAAAATACCTATTCCGGGCCGCCCAATACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 132) 931-i ATGAAAAAATACCTATTCCGGGCGGCTTTGTACGGGATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 123) 919sp ATGAAAAAATACCTATTCCGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 133) 9S1e MKKYLF......IAAAILAA (SEQ ID NO: 134) 9S1c MKKYLFRAAQYGIAAAILAA (SEQ ID NO: 135) 9S1b MKKYLFRAAQYGIAAAILAA (SEQ ID NO: 136) 9S1i MKKYLFRAALYGIAAAILAA (SEQ ID NO: 128) 919sp MKKYLFRAALYGIAAAILAA

As shown in the sequences alignments, most of the mutants analysed contain in-frame deletions which were unexpectedly produced by the host cells.

Selection of the mutants was performed by transforming E. coli BL21(DE3) cells with DNA prepared from a mixture of L1 and S1 mutated clones. Single transformants were screened for high PhoC activity by streaking them onto LB plates containing 100 μg/ml ampicillin, 50 μg/ml methyl green, 1 mg/ml PDP (phenolphthaleindiphosphate). On this medium PhoC-producing cells become green (FIG. 10).

A quantitative analysis of PhoC produced by these mutants was carried out in liquid medium using pNPP as a substrate for PhoC activity. The specific activities measured in cell extracts and supernatants of mutants grown in liquid medium for 0, 30, 90, 180 min. were:

Cell Extracts

0 30 90 180 control 0.00 0.00 0.00 0.00 9phoC 1.11 1.11 3.33 4.44 9S1e 102.12 111.00 149.85 172.05 9L1a 206.46 111.00 94.35 83.25 9L1d 5.11 4.77 4.00 3.11 9L1f 27.75 94.35 82.14 36.63 9S1b 156.51 111.00 72.15 28.86 9S1c 72.15 33.30 21.09 14.43 9S1i 156.51 83.25 55.50 26.64 phoCwt 194.25 180.93 149.85 142.08 Supernatants

0 30 90 180 control 0.00 0.00 0.00 0.00 9phoC 0.33 0.00 0.00 0.00 9S1e 0.11 0.22 0.44 0.89 9L1a 4.88 5.99 5.99 7.22 9L1d 0.11 0.11 0.11 0.11 9L1f 0.11 0.22 0.11 0.11 9S1b 1.44 1.44 1.44 1.67 9S1c 0.44 0.78 0.56 0.67 9S1i 0.22 0.44 0.22 0.78 phoCwt 34.41 43.29 87.69 177.60

Some of the mutants produce high amounts of PhoC and in particular, mutant 9L1a can secrete PhoC in the culture medium. This is noteworthy since the signal peptide sequence of this mutant is only 9 amino acids long. This is the shortest signal peptide described to date.

Example 21 C-Terminal Deletions of Maf-Related Proteins

MafB-related proteins include 730, ORF46 and ORF29.

The 730 protein from MC58 has the following sequence (SEQ ID NO:137):

1 VKPLRRLTNL LAACAVAAAA LIQPALAADL AQDPFITDNA QRQHYEPGGK 51 YHLFGDPRGS VSDRTGKINV IQDYTHQMGN LLIQQANING TIGYHTRFSG 101 HGHEEHAPFD NHAADSASEE KGNVDEGFTV YRLNWEGHEH HPADAYDGPK 151 GGNYPKPTGA RDEYTYHVNG TARSIKLNPT DTRSIRQRIS DNYSNLGSNF 201 SDRADEANRK MFEHNAKLDR WGNSMEFING VAAGALNPFI SAGEALGIGD 251 ILYGTRYAID KAAMRNIAPL PAEGKFAVIG GLGSVAGFEK NTREAVDRWI 301 QENPNAAETV EAVFNVAAAA KVAKLAKAAK PGKAAVSGDF ADSYKKKLAL 351 SDSARQLYQN AKYREALDIH YEDLIRRKTD GSSKFINGRE IDAVTNDALI 401 QAKRTISAID KPKNFLNQKN RKQIKATIEA ANQQGKRAEF WFKYGVHSQV 451 KSYIESKGGI VKTGLGD*

The leader peptide is underlined.

730 shows similar features to ORF46 (see example 8 above):

-   -   as for Orf46, the conservation of the 730 sequence among MenB,         MenA and gonococcus is high (>80%) only for the N-terminal         portion. The C-terminus, from ˜340, is highly divergent.     -   its predicted secondary structure contains a hydrophobic segment         spanning the central region of the molecule (aa. 227-247).     -   expression of the full-length gene in E. coli gives very low         yields of protein. Expression from tagged or untagged constructs         where the signal peptide sequence has been omitted has a toxic         effect on the host cells. In other words, the presence of the         full-length mature protein in the cytoplasm is highly toxic for         the host cell while its translocation to the periplasm (mediated         by the signal peptide) has no detectable effect on cell         viability. This “intracellular toxicity” of 730 is particularly         high since clones for expression of the leaderless 730 can only         be obtained at very low frequency using a recA genetic         background (E. coli strains: HB101 for cloning; HMS174(DE3) for         expression).

To overcome this toxicity, a similar approach was used for 730 as described in example 8 for ORF46. Four C-terminal truncated forms were obtained, each of which is well expressed. All were obtained from intracellular expression of His-tagged leaderless 730.

Form A consists of the N-terminal hydrophilic region of the mature protein (aa. 28-226). This was purified as a soluble His-tagged product, having a higher-than-expected MW.

Form B extends to the end of the region conserved between serogroups (aa. 28-340). This was purified as an insoluble His-tagged product.

The C-terminal truncated forms named C1 and C2 were obtained after screening for clones expressing high levels of 730-His clones in strain HMS174(DE3). Briefly, the pET21b plasmid containing the His-tagged sequence coding for the full-length mature 730 protein was used to transform the recA strain HMS174(DE3). Transformants were obtained at low frequency which showed two phenotypes: large colonies and very small colonies. Several large and small colonies were analysed for expression of the 730-His clone. Only cells from large colonies over-expressed a protein recognised by anti-730A antibodies. However the protein over-expressed in different clones showed differences in molecular mass. Sequencing of two of the clones revealed that in both cases integration of an E. coli IS sequence had occurred within the sequence coding for the C terminal region of 730. The two integration events have produced in-frame fusion with 1 additional codon in the case of C1, and 12 additional codons in the case of C2 (FIG. 11). The resulting “mutant” forms of 730 have the following sequences:

730-C1 (due to an IS1 insertion-FIG. 11A) (SEQ ID NO: 138) 1 MADLAQDPFI TDNAQRQHYE PGGKYHLFGD PRGSVSDRTG KINVIQDYTH 51 QMGNLLIQQA NINGTIGYHT RFSGHGHEEH APFDNHAADS ASEEKGNVDE 101 GFTVYRLNWE GHEHHPADAY DGPKGGNYPR PTGARDEYTY HVNGTARSIK 151 LNPTDTRSIR QRISDNYSNL GSNFSDRADE ANRKMFEHNA KLDRWGNSME 201 FINGVAAGAL NPFISAGEAL GIGDILYGTR YAIDKAAMRN IAPLPAEGEF 251 AVIGGLGSVA GFEKNTREAV DRWIQENPNA AETVEAVFNV AAAAKVAKLA 301 KAAKPGKAAV SGDFADSYKK KLALSDSARQ LYQNAKYREA LDIHYEDLIR 351 RKTDGSSKFI NGREIDAVTN DALIQAR*

The additional amino acid produced by the insertion is underlined.

730-C2 (due to an IS5 insertion-FIG. 11B) (SEQ ID NO: 139) 1 MADLAQDPFI TDNAQRQHYE PGGKYHLFGD PRGSVSDRTG KINVIQDYTH 51 QMGNLLIQQA NINGTIGYHT RFSGHGHEEH APFDNHAADS ASEEKGNVDE 101 GFTVYRLNWE GHEHHPADAY DGPKGGNYPK PTGARDEYTY HVNGTARSIK 151 LNPTDTRSIR QRISDNYSNL GSNFSDRADE ANRKMFEHNA KLDRWGNSME 201 FINGVAAGAL NPFISAGEAL GIGDILYGTR YAIDKAAMRN IAPLPAEGKF 251 AVIGGLGSVA GFEKNTREAV DRWIQENPNA AETVEAVFNV AAAAKVAKLA 301 KAAKPGKAAV SGDFADSYKK KLALSDSARQ LYQNAKYREA LGKVRISGEI 351 LLG*

The additional amino acids produced by the insertion are underlined.

In conclusion, intracellular expression of the 730-C1 form gives very high level of protein and has no toxic effect on the host cells, whereas the presence of the native C-terminus is toxic. These data suggest that the “intracellular toxicity” of 730 is associated with the C-terminal 65 amino acids of the protein.

Equivalent truncation of ORF29 to the first 231 or 368 amino acids has been performed, using expression with or without the leader peptide (amino acids 1-26; deletion gives cytoplasmic expression) and with or without a His-tag.

Example 22 Domains in 961

As described in example 9 above, the GST-fusion of 961 was the best-expressed in E. coli. To improve expression, the protein was divided into domains (FIG. 12).

The domains of 961 were designed on the basis of YadA (an adhesin produced by Yersinia which has been demonstrated to be an adhesin localized on the bacterial surface that forms oligomers that generate surface projection [Hoiczyk et al. (2000) EMBO J. 19:5989-99]) and are: leader peptide, head domain, coiled-coil region (stalk), and membrane anchor domain.

These domains were expressed with or without the leader peptide, and optionally fused either to C-terminal His-tag or to N-terminal GST. E. coli clones expressing different domains of 961 were analyzed by SDS-PAGE and western blot for the production and localization of the expressed protein, from over-night (o/n) culture or after 3 hours induction with IPTG. The results were:

Total lysate Periplasm (Western (Western Supernatant OMV Blot) Blot) (Western Blot) SDS-PAGE 961 (o/n) − − − 961 (IPTG) +/− − − 961-L (o/n) + − − + 961-L (IPTG) + − − + 961c-L (o/n) − − − 961c-L (IPTG) + + + 961Δ₁-L (o/n) − − − 961Δ₁-L (IPTG) + − − +

The results show that in E. coli:

-   -   961-L is highly expressed and localized on the outer membrane.         By western blot analysis two specific bands have been detected:         one at ˜45 kDa (the predicted molecular weight) and one at ˜180         kDa, indicating that 961-L can form oligomers. Additionally,         these aggregates are more expressed in the over-night culture         (without IPTG induction). OMV preparations of this clone were         used to immunize mice and serum was obtained. Using overnight         culture (predominantly by oligomeric form) the serum was         bactericidal; the IPTG-induced culture (predominantly monomeric)         was not bactericidal.     -   961Δ₁-L (with a partial deletion in the anchor region) is highly         expressed and localized on the outer membrane, but does not form         oligomers;     -   the 961c-L (without the anchor region) is produced in soluble         form and exported in the supernatant.

Titres in ELISA and in the serum bactericidal assay using His-fusions were as follows:

ELISA Bactericidal 961a (aa 24-268) 24397 4096 961b (aa 269-405) 7763 64 961c-L 29770 8192 961c (2996) 30774 >65536 961c (MC58) 33437 16384 961d 26069 >65536

E. coli clones expressing different forms of 961 (961, 961-L, 961Δ₁-L and 961c-L) were used to investigate if the 961 is an adhesin (c.f. YadA). An adhesion assay was performed using (a) the human epithelial cells and (b) E. coli clones after either over-night culture or three hours IPTG induction. 961-L grown over-night (961Δ₁-L) and IPTG-induced 961c-L (the clones expressing protein on surface) adhere to human epithelial cells.

961c was also used in hybrid proteins (see above). As 961 and its domain variants direct efficient expression, they are ideally suited as the N-terminal portion of a hybrid protein.

Example 23 Further Hybrids

Further hybrid proteins of the invention are shown below (see also FIG. 14). These are advantageous when compared to the individual proteins:

ORF46.1-741 (SEQ ID NOs: 140 and 141) 1 ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA 51 GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC 101 TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG 151 TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA 201 CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA 251 ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA 301 TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA 351 CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA 401 GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC 451 AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGCATCCA 501 CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG 551 CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC 601 TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA 651 AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA 701 TTGCTGTCAT GCACCGCTCG GGTCTGCTTT CCACCGAAAA CAAGATGGCG 751 CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC 801 AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG 851 AAGCCGTCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATTGGA 901 GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA 951 GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA 1001 GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT 1051 TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT 1101 CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG 1151 ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG 1201 AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GGGGTGGTGT 1251 CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA TGCACTAACC GCACCGCTCG 1301 ACCATAAAGA CAAAGGTTTG CAGTCTTTGA CGCTGGATCA GTCCGTCAGG 1351 AAAAACGAGA AACTGAAGCT GGCGGCACAA GGTGCGGAAA AAACTTATGG 1401 AAACGGTGAC AGCCTCAATA CGGGCAAATT GAAGAACGAC AAGGTCAGCC 1451 GTTTCGACTT TATCCGCCAA ATCGAAGTGG ACGGGCAGCT CATTACCTTG 1501 GAGAGTGGAG AGTTCCAAGT ATACAAACAA AGCCATTCCG CCTTAACCGC 1551 CTTTCAGACC GAGCAAATAC AAGATTCGGA GCATTCCGGG AAGATGGTTG 1601 CGAAACGCCA GTTCAGAATC GGCGACATAG CGGGCGAACA TACATCTTTT 1651 GACAAGCTTC CCGAAGGCGG CAGGGCGACA TATCGCGGGA CGGCGTTCGG 1701 TTCAGACGAT GCCGGCGGAA AACTGACCTA CACCATAGAT TTCGCCGCCA 1751 AGCAGGGAAA CGGCAAAATC GAACATTTGA AATCGCCAGA ACTCAATGTC 1801 GACCTGGCCG CCGCCGATAT CAAGCCGGAT GGAAAACGCC ATGCCGTCAT 1851 CAGCGGTTCC GTCCTTTACA ACCAAGCCGA GAAAGGCAGT TACTCCCTCG 1901 GTATCTTTGG CGGAAAAGCC CAGGAAGTTG CCGGCAGCGC GGAAGTGAAA 1951 ACCGTAAACG GCATACGCCA TATCGGCCTT GCCGCCAAGC AACTCGAGCA 2001 CCACCACCAC CACCACTGA 1 MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ 51 LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG 101 FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL 151 NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA 201 FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA 251 RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG 301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH 351 SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP 401 NFEKHVKYDT GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR 451 KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL 501 ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF 551 DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV 601 DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK 651 TVNGIRHIGL AAKQLEHHHH HH* ORF46.1-961 (SEQ ID NOs: 142 and 143) 1 ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA 51 GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC 101 TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG 151 TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA 201 CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA 251 ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA 301 TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA 351 CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA 401 GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC 451 AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGTTTCCA 501 CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG 551 CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC 601 TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA 651 AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA 701 TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG 751 CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC 801 AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG 851 AAGCCGTCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATTGGA 901 GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA 951 GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GGAAGTGAAA TCCGCCGTCA 1001 GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT 1051 TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT 1101 CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG 1151 ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG 1201 AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GAGGAGGAGC 1251 CACAAACGAC GACGATGTTA AAAAAGCTGC CACTGTGGCC ATTGCTGCTG 1301 CCTACAACAA TGGCCAAGAA ATCAACGGTT TCAAAGCTGG AGAGACCATC 1351 TACGACATTG ATGAAGACGG CACAATTACC AAAAAAGACG CAACTGCAGC 1401 CGATGTTGAA GCCGACGACT TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA 1451 CTAACCTGAC CAAAACCGTC AATGAAAACA AACAAAACGT CGATGCCAAA 1501 GTAAAAGCTG CAGAATCTGA AATAGAAAAG TTAACAACCA AGTTAGCAGA 1551 CACTGATGCC GCTTTAGCAG ATACTGATGC CGCTCTGGAT GCAACCACCA 1601 ACGCCTTGAA TAAATTGGGA GAAAATATAA CGACATTTGC TGAAGAGACT 1651 AAGACAAATA TCGTAAAAAT TGATGAAAAA TTAGAAGCCG TGGCTGATAC 1701 CGTCGACAAG CATGCCGAAG CATTCAACGA TATCGCCGAT TCATTGGATG 1751 AAACCAACAC TAAGGCAGAC GAAGCCGTCA AAACCGCCAA TGAAGCCAAA 1801 CAGACGGCCG AAGAAACCAA ACAAAACGTC GATGCCAAAG TAAAAGCTGC 1851 AGAAACTGCA GCAGGCAAAG CCGAAGCTGC CGCTGGCACA GCTAATACTG 1901 CAGCCGACAA GGCCGAAGCT GTCGCTGCAA AAGTTACCGA CATCAAAGCT 1951 GATATCGCTA CGAACAAAGA TAATATTGCT AAAAAAGCAA ACAGTGCCGA 2001 CGTGTACACC AGAGAAGAGT CTGACAGCAA ATTTGTCAGA ATTGATGGTC 2051 TGAACGCTAC TACCGAAAAA TTGGACACAC GCTTGGCTTC TGCTGAAAAA 2101 TCCATTGCCG ATCACGATAC TCGCCTGAAC GGTTTGGATA AAACAGTGTC 2151 AGACCTGCGC AAAGAAACCC GCCAAGGCCT TGCAGAACAA GCCGCGCTCT 2201 CCGGTCTGTT CCAACCTTAC AACGTGGGTC GGTTCAATGT AACGGCTGCA 2251 GTCGGCGGCT ACAANTCCGA ATCGGCAGTC GCCATCGGTA CCGGCTTCCG 2301 CTTTACCGAA AACTTTGCCG CCAAAGCAGG CGTGGCAGTC GGCACTTCGT 2351 CCGGTTCTTC CGCAGCCTAC CATGTCGGCG TCAATTACGA GTGGCTCGAG 2401 CACCACCACC ACCACCACTG A 1 MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ 51 LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG 101 FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL 151 NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA 201 FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA 251 RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG 301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH 351 SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP 401 NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI 451 YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK 501 VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET 551 KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK 601 QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA 651 DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK 701 SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGRFNVTAA 751 VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY HVGVNYEWLE 801 HHHHHH* ORF46.1-961c (SEQ ID NOs: 144 and 145) 1 ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA 51 GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC 101 TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG 151 TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA 201 CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA 251 ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA 301 TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA 351 CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA 401 GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC 451 AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGTTTCCA 501 CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG 551 CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC 601 TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA 651 AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA 701 TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG 751 CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC 801 AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG 851 AAGCCATCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATTGGA 901 GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA 951 GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA 1001 GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT 1051 TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT 1101 CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG 1151 ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG 1201 AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GAGGAGGAGC 1251 CACAAACGAC GACGATGTTA AAAAAGCTGC CACTGTGGCC ATTGCTGCTG 1301 CCTACAACAA TGGCCAAGAA ATCAACGGTT TCAAAGCTGG AGAGACCATC 1351 TACGACATTG ATGAAGACGG CACAATTACC AAAAAAGACG CAACTGCAGC 1401 CGATGTTGAA GCCGACGACT TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA 1451 CTAACCTGAC CAAAACCGTC AATGAAAACA AACAAAACGT CGATGCCAAA 1501 GTAAAAGCTG CAGAATCTGA AATAGAAAAG TTAACAACCA AGTTAGCAGA 1551 CACTGATGCC GCTTTAGCAG ATACTGATGC CGCTCTGGAT GCAACCACCA 1601 ACGCCTTGAA TAAATTGGGA GAAAATATAA CGACATTTGC TGAAGAGACT 1651 AAGACAAATA TCGTAAAAAT TGATGAAAAA TTAGAAGCCG TGGCTGATAC 1701 CGTCGACAAG CATGCCGAAG CATTCAACGA TATCGCCGAT TCATTGGATG 1751 AAACCAACAC TAAGGCAGAC GAAGCCGTCA AAACCGCCAA TGAAGCCAAA 1801 CAGACGGCCG AAGAAACCAA ACAAAACGTC GATGCCAAAG TAAAAGCTGC 1851 AGAAACTGCA GCAGGCAAAG CCGAAGCTGC CGCTGGCACA GCTAATACTG 1901 CAGCCGACAA GGCCGAAGCT GTCGCTGCAA AAGTTACCGA CATCAAAGCT 1951 GATATCGCTA CGAACAAAGA TAATATTGCT AAAAAAGCAA ACAGTGCCGA 2001 CGTGTACACC AGAGAAGAGT CTGACAGCAA ATTTGTCAGA ATTGATGGTC 2051 TGAACGCTAC TACCGAAAAA TTGGACACAC GCTTGGCTTC TGCTGAAAAA 2101 TCCATTGCCG ATCACGATAC TCGCCTGAAC GGTTTGGATA AAACAGTGTC 2151 AGACCTGCGC AAAGAAACCC GCCAAGGCCT TGCAGAACAA GCCGCGCTCT 2201 CCGGTCTGTT CCAACCTTAC AACGTGGGTC TCGAGCACCA CCACCACCAC 2251 CACTGA 1 MSDLANDSFI RQVLDRQHFE PDGKYKLFGS RGELAERSGH IGLGKIQSHQ 51 LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG 101 FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL 151 NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA 201 FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA 251 RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG 301 AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH 351 SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP 401 NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI 451 YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK 501 VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET 551 KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK 601 QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA 651 DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK 701 SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGLEHHHHH 751 H* 961-ORF46.1 (SEQ ID NOs: 146 and 147) 1 ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC 51 TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGATTCAAA GCTGGAGAGA 101 CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT 151 GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT 201 CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG 251 CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA 301 GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC 351 CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG 401 AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT 451 GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT 501 GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG 551 CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA 601 GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA 651 TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA 701 AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT 751 GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA 801 TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG 851 AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA 901 GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC 951 GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG 1001 CTGCAGTCGG CGOCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC 1051 TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC 1101 TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG 1151 GATCCGGAGG AGGAGGATCA GATTTGGCAA ACGATTCTTT TATCCGGCAG 1201 GTTCTCGACC GTCAGCATTT CGAACCCGAC GGGAAATACC ACCTATTCGG 1251 CAGCAGGGGG GAACTTGCCG AGCGCAGCGG CCATATCGGA TTGGGAAAAA 1301 TACAAAGCCA TCAGTTGGGC AACCTGATGA TTCAACAGGC GGCCATTAAA 1351 GGAAATATCG GCTACATTGT CCGCTTTTCC GATCACGGGC ACGAAGTCCA 1401 TTCCCCCTTC GACAACCATG CCTCACATTC CGATTCTGAT GAAGCCGGTA 1451 GTCCCGTTGA CGGATTTAGC CTTTACCGCA TCCATTGGGA CGGATACGAA 1501 CACCATCCCG CCGACGGCTA TGACGGGCCA CAGGGCGGCG GCTATCCCGC 1551 TCCCAAAGGC GCGAGGGATA TATACAGCTA CGACATAAAA GGCGTTGCCC 1601 AAAATATCCG CCTCAACCTG ACCGACAACC GCAGCACCGG ACAACGGCTT 1651 GCCGACCGTT TCCACAATGC CGGTAGTATG CTGACGCAAG GAGTAGGCGA 1701 CGGATTCAAA CGCGCCACCC GATACAGCCC CGAGCTGGAC AGATCGGGCA 1751 ATGCCGCCGA AGCCTTCAAC GGCACTGCAG ATATCGTTAA AAACATCATC 1801 GGCGCGGCAG GAGAAATTGT CGGCGCAGGC GATGCCGTGC AGGGCATAAG 1851 CGAAGGCTCA AACATTGCTG TCATGCACGG CTTGGGTCTG CTTTCCACCG 1901 AAAACAAGAT GGCGCGCATC AACGATTTGG CAGATATGGC GCAACTCAAA 1951 GACTATGCCG CAGCAGCCAT CCGCGATTGG GCAGTCCAAA ACCCCAATGC 2001 CGCACAAGGC ATAGAAGCCG TCAGCAATAT CTTTATGGCA GCCATCCCCA 2051 TCAAAGGGAT TGGAGCTGTT CGGGGAAAAT ACGGCTTGGG CGGCATCACG 2101 GCACATCCTA TCAAGCGGTC GCAGATGGGC GCGATCGCAT TGCCGAAAGG 2151 GAAATCCGCC GTCAGCGACA ATTTTGCCGA TGCGGCATAC GCCAAATACC 2201 CGTCCCCTTA CCATTCCCGA AATATCCGTT CAAACTTGGA GCAGCGTTAC 2251 GGCAAAGAAA ACATCACCTC CTCAACCGTG CCGCCGTCAA ACGGCAAAAA 2301 TGTCAAACTG GCAGACCAAC GCCACCCGAA GACAGGCGTA CCGTTTGACG 2351 GTAAAGGGTT TCCGAATTTT GAGAAGCACG TGAAATATGA TACGCTCGAG 2401 CACCACCACC ACCACCACTG A 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL 101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 151 DTVDEHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TYQNVDAKVK 201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT 301 VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG 351 FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGS DLANDSFIRQ 401 VLDRQHFEPD GKEHLFGSRG ELAERSGHIG LGKIQSHQLG NLMIQQAAIK 451 GNIGYIVRFS DHGHEVHSPF DNHASHSDSD EAGSPVDGFS LYRIHWDGYE 501 HHPADGYDGP QGGGYPAPKG ARDIYSYDEK GVAQNIRLNL TDNRSTGQRL 551 ADRFHNAGSM LTQGVGDGFK RATRYSPELD RSGNAAEAFN GTADIVKNII 601 GAAGEIVGAG DAVQGISEGS NIAVMHGLGL LSTENKMARI NDLADMAQLK 651 DYAAAAIRDW AVQNPNAAQG IEAVSNIFMA AIPIKGIGAV RGKYGLGGIT 701 AHPIKRSQMG AIALPKGKSA VSDNFADAAY AKYPSPYHSR NIRSNLEQRY 751 GKENITSSTV PPSNGKNVKL ADQRHPKTGV PFDGKGFPNF EKHVKYDTLE 801 HHHHHH* 961-741 (SEQ ID NOs: 148 and 149) 1 ATGGCCACAA ACGACGACGA TGTTAAAAAA GCAGACACTG TGGCCATTGC 51 TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA 101 CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT 151 GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT 201 CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG 251 CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA 301 GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC 351 CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG 401 AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT 451 GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT 501 GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG 551 CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA 601 GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA 651 TACTGCAGCC GACTATGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA 701 AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT 751 GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA 801 TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG 851 AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA 901 GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC 951 GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG 1001 CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC 1051 TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC 1101 TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG 1151 GATCCGGAGG GGGTGGTGTC GCCGCCGACA TCGGTGCGGG GCTTGCCGAT 1201 GCACTAACCG CACCGCTCGA CCATAAAGAC AAAGGTTTGC AGTCTTTGAC 1251 GCTGGATCAG TCCGTCAGGA AAAACGAGAA ACTGAAGCTG GCGGCACAAG 1301 GTGCGGAAAA AACTTATGGA AACGGTGACA GCCTCAATAC GGGCAAATTG 1351 AAGAACGACA AGGTCAGCCG TTTCGACTTT ATCCGCCAAA TCGAAGTGGA 1401 CGGGCAGCTC ATTACCTTGG AGAGTGGAGA GTTCCAAGTA TACAAACAAA 1451 GCCATTCCGC CTTAACCGCC TTTCAGACCG AGCAAATACA AGATTCGGAG 1501 CATTCCGGGA AGATGGTTGC GAAACGCCAG TTCAGAATCG GCGACATAGC 1551 GGGCGAACAT ACATCTTTTG ACAAGCATGC CGAAGGCGGC AGGGCGACAT 1601 ATCGCGGGAC GGCGTTCGGT TCAGACGATG CCGGCGGAAA ACTGACCTAC 1651 ACCATAGATT TCGCCGCCAA GCAGGGAAAC GGCAAAATCG AACATTTGAA 1701 ATCGCCAGAA CTCAATGTCG ACCTGGCCGC CGCCGATATC AAGCCGGATG 1751 GAAAACGCCA TGCCGTCATC AGCGGTTCCG TCCTTTACAA CCAAGCCGAG 1801 AAAGGCAGTT ACTCCCTCGG TATCTTTGGC GGAAAAGCCC AGGAAGTTGC 1851 CGGCAGCGCG GAAGTGAAAA CCGTAAACGG CATACGCCAT ATCGGCCTTG 1901 CCGCCAAGCA ACTCGAGCAC CACCACCACC ACCACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL 101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 151 DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK 201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT 301 VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG 351 FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGV AADIGAGLAD 401 ALTAPLDHKD KGLQSLTLDQ SVRKNEKLKL AAQGAEKTYG NGDSLNTGKL 451 KNDKVSRFDF IRQIEVDGQL ITLESGEFQV YKQSHSALTA FQTEQIQDSE 501 HSGKMVAKRQ FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY 551 TIDFAAKQGN GKIEHLKSPE LNVDLAAADI KPDGKRHAVI SGSVLYNQAE 601 KGSYSLGIFG GKAQEVAGSA EVKTVNGIRH IGLAAKQLEH HHHHH* 961-983 (SEQ ID NOs: 150 and 151) 1 ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC 51 TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA 101 CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT 151 GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT 201 CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG 251 CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA 301 GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC 351 CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG 401 AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT 451 GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT 501 GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG 551 CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA 601 GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA 651 TACTCCACCC CACAAGGCCG AAGCTGTCCC TCCAAAACTT ACCOACATCA 701 AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT 751 GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA 801 TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG 851 AAAAAGGCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA 901 GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC 951 GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG 1001 CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC 1051 TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC 1101 TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG 1151 GATCCGGCGG AGGCGGCACT TCTGCGCCCG ACTTCAATGC AGGCGGTACC 1201 GGTATCGGCA GCAACAGCAG AGCAACAACA GCGAAATCAG CAGCAGTATC 1251 TTACGCCGGT ATCAAGAACG AAATGTGCAA AGACAGAAGC ATGCTCTGTG 1301 CCGGTCGGGA TGACGTTGCG GTTACAGACA GGGATGCCAA AATCAATGCC 1351 CCCCCCCCGA ATCTGCATAC CGGAGACTTT CCAAACCCAA ATGACGCATA 1401 CAAGAATTTG ATCAACCTCA AACCTGCAAT TGAAGCAGGC TATACAGGAC 1451 GCGGGGTAGA GGTAGGTATC GTCGACACAG GCGAATCCGT CGGCAGCATA 1501 TCCTTTCCCG AACTGTATGG CAGAAAAGAA CACGGCTATA ACGAAAATTA 1551 CAAAAACTAT ACGGCGTATA TGCGGAAGGA AGCGCCTGAA GACGGAGGCG 1601 GTAAAGACAT TGAAGCTTCT TTCGACGATG AGGCCGTTAT AGAGACTGAA 1651 GCAAAGCCGA CGGATATCCG CCACGTAAAA GAAATCGGAC ACATCGATTT 1701 GGTCTCCCAT ATTATTGGCG GGCGTTCCGT GGACGGCAGA CCTGCAGGCG 1751 GTATTGCGCC CGATGCGACG CTACACATAA TGAATACGAA TGATGAAACC 1801 AAGAACGAAA TGATGGTTGC AGCCATCCGC AATGCATGGG TCAAGCTGGG 1851 CGAACGTGGC GTGCGCATCG TCAATAACAG TTTTGGAACA ACATCGAGGG 1901 CAGGCACTGC CGACCTTTTC CAAATAGCCA ATTCGGAGGA GCAGTACCGC 1951 CAAGCGTTGC TCGACTATTC CGGCGGTGAT AAAACAGACG AGGGTATCCG 2001 CCTGATGCAA CAGAGCGATT ACGGCAACCT GTCCTACCAC ATCCGTAATA 2051 AAAACATGCT TTTCATCTTT TCGACAGGCA ATGACGCACA AGCTCAGCCC 2101 AACACATATG CCCTATTGCC ATTTTATGAA AAAGACGCTC AAAAAGGCAT 2151 TATCACAGTC GCAGGCGTAG ACCGCAGTGG AGAAAAGTTC AAACGGGAAA 2201 TGTATGGAGA ACCGGGTACA GAACCGCTTG AGTATGGCTC CAACCATTGC 2251 GGAATTACTG CCATGTGGTG CCTGTCGGCA CCCTATGAAG CAAGCGTCCG 2301 TTTCACCCGT ACAAACCCGA TTCAAATTGC CGGAACATCC TTTTCCGCAC 2351 CCATCGTAAC CGGCACGGCG GCTCTGCTGC TGCAGAAATA CCCGTGGATG 2401 AGCAACGACA ACCTGCGTAC CACGTTGCTG ACGACGGCTC AGGACATCGG 2451 TGCAGTCGGC GTGGACAGCA AGTTCGGCTG GGGACTGCTG GATGCGGGTA 2501 AGGCCATGAA CGGACCCGCG TCCTTTCCGT TCGGCGACTT TACCGCCGAT 2551 ACGAAAGGTA CATCCGATAT TGCCTACTCC TTCCGTAACG ACATTTCAGG 2601 CACGGGCGGC CTGATCAAAA AAGGCGGCAG CCAACTGCAA CTGCACGGCA 2651 ACAACACCTA TACGGGCAAA ACCATTATCG AAGGCGGTTC GCTGGTGTTG 2701 TACGGCAACA ACAAATCGGA TATGCGCGTC GAAACCAAAG GTGCGCTGAT 2751 TTATAACGGG GCGGCATCCG GCGGCAGCCT GAACAGCGAC GGCATTGTCT 2801 ATCTGGCAGA TACCGACCAA TCCGGCGCAA ACGAAACCGT ACACATCAAA 2851 GGCAGTCTGC AGCTGGACGG CAAAGGTACG CTGTACACAC GTTTGGGCAA 2901 ACTGCTGAAA GTGGACGGTA CGGCGATTAT CGGCGGCAAG CTGTACATGT 2951 CGGCACGCGG CAAGGGGGCA GGCTATCTCA ACAGTACCGG ACGACGTGTT 3001 CCCTTCCTGA GTGCCGCCAA AATCGGGCAG GATTATTCTT TCTTCACAAA 3051 CATCGAAACC GACGGCGGCC TGCTGGCTTC CCTCGACAGC GTCGAAAAAA 3101 CAGCGGGCAG TGAAGGCGAC ACGCTGTCCT ATTATGTCCG TCGCGGCAAT 3151 GCGGCACGGA CTGCTTCGGC AGCGGCACAT TCCGCGCCCG CCGGTCTGAA 3201 ACACGCCGTA GAACAGGGCG GCAGCAATCT GGAAAACCTG ATGGTCGAAC 3251 TGGATGCCTC CGAATCATCC GCAACACCCG AGACGGTTGA AACTGCGGCA 3301 GCCGACCGCA CAGATATGCC GGGCATCCGC CCCTACGGCG CAACTTTCCG 3351 CGCAGCGGCA GCCGTACAGC ATGCGAATGC CGCCGACGGT GTACGCATCT 3401 TCAACAGTCT CGCCGCTACC GTCTATGCCG ACAGTACCGC CGCCCATGCC 3451 GATATGCAGG GACGCCGCCT GAAAGCCGTA TCGGACGGGT TGGACCACAA 3501 CGGCACGGGT CTGCGCGTCA TCGCGCAAAC CCAACAGGAC GGTGGAACGT 3551 GGGAACAGGG CGGTGTTGAA GGCAAAATGC GCGGCAGTAC CCAAACCGTC 3601 GGCATTGCCG CGAAAACCGG CGAAAATACG ACAGCAGCCG CCACACTGGG 3651 CATGGGACGC AGCACATGGA GCGAAAACAG TGCAAATGCA AAAACCGACA 3701 GCATTAGTCT GTTTGCAGGC ATACGGCACG ATGCGGGCGA TATCGGCTAT 3751 CTCAAAGGCC TGTTCTCCTA CGGACGCTAC AAAAACAGCA TCAGCCGCAG 3801 CACCGGTGCG GACGAACATG CGGAAGGCAG CGTCAACGGC ACGCTGATGC 3851 AGCTGGGCGC ACTGGGCGGT GTCAACGTTC CGTTTGCCGC AACGGGAGAT 3901 TGAACGGACG AAGGCGGTCT GCGCTACGAC CTGCTCAAAC AGGATGCATT 3951 CGCCGAAAAA GGCAGTGCTT TGGGCTGGAG CGGCAACAGC CTCACTGAAG 4001 GCACGCTGGT CGGACTCGCG GGTCTGAAGC TGTCGCAACC CTTGAGCGAT 4051 AAAGCCGTCC TGTTTGCAAC GGCGGGCGTG GAACGCGACC TGAACGGACG 4101 CGACTACACG GTAACGGGCG GCTTTACCGG CGCGACTGCA GCAACCGGCA 4151 AGACGGGGGC ACGCAATATG CCGCACACCC GTCTGGTTGC CGGCCTGGGC 4201 GCGGATGTCG AATTCGGCAA CGGCTGGAAC GGCTTGGCAC GTTACAGCTA 4251 CGCCGGTTCC AAACAGTACG GAAACCAAAG CGGACGAGTC GGCGTAGGCT 4301 ACCGGTTCCT CGAGCACCAC CACCACCACC ACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL 101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 151 DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK 201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT 301 VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG 351 FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGT SAPDFNAGGT 401 GIGSNSRATT AKSAAVSYAG IKNEMCKDRS MLCAGRDDVA VTDRDAKINA 451 PPPNLHTGDF PNPNDAYKNL INLKPAIEAG YTGRGVEVGI VDTGESVGSI 501 SFPELYGRKE HGYNENYKNY TAYMRKEAPE DGGGKDIEAS FDDEAVIETE 551 AKPTDIRHVK EIGHIDLVSH IIGGRSVDGR PAGGIAPDAT LHIMNTNDET 601 KNEMMVAAIR NAWVKLGERG VRIVNNSFGT TSRAGTADLF QIANSEEQYR 651 QALLDYSGGD KTDEGIRLMQ QSDYGNLSYH IRNKNMLFIF STGNDAQAQP 701 NTYALLPFYE KDAQKGIITV AGVDRSGEKF KREMYGEPGT EPLEYGSNHC 751 GITAMWCLSA PYEASVRFTR TNPIQIAGTS FSAPIVTGTA ALLLQKYPWM 801 SNDNLRTTLL TTAQDIGAVG VDSKFGWGLL DAGKAMNGPA SFPFGDFTAD 851 TKGTSDIAYS FRNDISGTGG LIKKGGSQLQ LHGNNTYTGK TIIEGGSLVL 901 YGNNKSDMRV ETKGALIYNG AASGGSLNSD GIVYLADTDQ SGANETVHIK 951 GSLQLDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGKGA GYLNSTGRRV 1001 PFLSAAKIGQ DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSYYVRRGN 1051 AARTASAAAH SAPAGLKHAV EQGGSNLENL MVELDASESS ATPETVETAA 1101 ADRTDMPGIR PYGATFRAAA AVQHANAADG VRIFNSLAAT VYADSTAAHA 1151 DMOGRRLEAV SDGLDENGTG LRVIAQTQQD GGTWEQGGVE GKMRGSTQTV 1201 GIAAKTGENT TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY 1251 LKGLFSYGRY KNSISRSTGA DEHAEGSVNG TLMQLGALGG VNVPFAATGD 1301 LTVEGGLRYD LLKQDAFAEK GSALGWSGNS LTEGTLVGLA GLKLSQPLSD 1351 KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG 1401 ADVEFGNGWN GLARYSYAGS KQYGNHSGRV GVGYRFLEHH HHHH* 961c-ORF46.1 (SEQ ID NOs: 152 and 153) 1 ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC 51 TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA 101 CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT 151 GCTGCCGCTG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT 201 CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG 251 CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA 301 GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC 351 CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG 401 AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATGTCA AGCCGTGGCT 451 GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT 501 GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG 551 CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA 601 GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA 651 TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGAAAAAAGT ACCGACATCA 701 AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT 751 GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA 801 TGGTCTGAAC GCTACTACCG AAAAATGTCA CACACOCTTG GCTTCTGCTG 851 AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA 901 GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC 951 GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGAGGAGGAG 1001 GATCAGATTT GGCAAACGAT TCTTTTATCC GGCAGGTTCT CGACCGTCAG 1051 CATTTCGAAC CCGACGGGAA ATACCACCTA TTGGGCGGCA GGGGGGAACT 1101 TGCCGAGCGC AGCGGCCATA TCGGATTGGG AAAAATACAA AGCCATCAGT 1151 TGGGCAACCT GATGATTCAA CAGGCGGCCA TTAAAGGAAA TATCGGCTAC 1201 ATTGTCCGCT TTTCCGATCA CGGGCACGAA GTCCATTCCC CCTTCGACAA 1251 CCATGCCTCA CATTCCGATT CTGATGAAGC CGGTAGTCCC GTTGACOGAT 1301 TTAGCCTTTA CCGCATCCAT TGGGACGGAT ACGAACACCA TCCCGCCGAC 1351 GGCTATGACG GGCCACAGGG CGGCGGCTAT CCCGCTCCCA AAGGCGCGAG 1401 GGATATATAC AGCTACGACA TAAAAGGCGT TGCCCAAAAT ATCCGCCTCA 1451 ACCTGACCGA CAACCGCAGC ACCGGACAAC GGCTTGCCGA CCGTTTCCAC 1501 AATGCCGGTA GTATGCTGAC GCAAGGAGTA GGCGACGGAT TCAAACGCGC 1551 CACCCGATAC AGCCCCGAGc TGGACAGATC GGGCAATGCC GCCGAAGCCT 1601 TCAACGGCAC TGCAGATATC GTTAAAAACA TCATCGGCGC GGCAGGAGAA 1651 ATTGTCGGCG CAGGCGATGC CGTGCAGGGC ATAAGCGAAG GCTCAAACAT 1701 TGCTGTCATG CACGGCTTGG GTCTGCTTTC CACCGAAAAC AAGATGGCGC 1751 GCATCAACGA TTTGGCAGAT ATGGCGCAAC TCAAAGACTA TGcCGCAGCA 1801 GCCATCCGCG ATTGGGCAGT CCAAAACCCC AATGCCGCAC AAGGCATAGA 1851 AGCCGTCAGC AATATCTTTA TGGCAGCCAT CCCCATCAAA GGGATTGGAG 1901 CTGTTCGGGG AAAATACGGC TTGGGCGGCA TCACGGCACA TCCTATCAAG 1951 CGGTCGCAGA TGGGCGCGAT CGCATTGCCG AAAGGGAAAT CCGCCGTCAG 2001 CGACAATTTT GCCGATGCGG CATACGCCAA ATACCCGTCC CCTTACCATT 2051 CCCGAAATAT CCGTTCAAAC TTGGAGCAGC GTTACGGCAA AGAAAACATC 2101 ACCTCCTCAA CCGTGCCGCC GTCAAACGGC AAAAATGTCA AACTGGCAGA 2151 CCAACGCCAC CCGAAGACAG GCGTACCGTT TGACGGTAAA GGGTTTCCGA 2201 ATTTTGAGAA GCACGTGAAA TATGATACGC TCGAGCACCA CCACCACCAC 2251 CACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL 101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 151 DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK 201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT 301 VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGSDLAND SFIRQVLDRQ 351 HFEPDGKYHL FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY 401 IVRFSDHGNE VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD 451 GYDGPQGGGY PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH 501 NAGSMLTQGV GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE 551 IVGAGDAVQG ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA 601 AIRDWAVQNP NAAQGIEAVS NIFNAAIPIK GIGAVRGKYG LGGITAHPIK 651 RSQMGAIALP KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI 701 TSSTVPPSNG KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTLEHHHHH 751 H* 961c-741 (SEQ ID NOs: 154 and 155) 1 ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC 51 TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA 101 CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT 151 GCAGCCGATG TTGAAGCCGA CGACTTLAAA GGTCTGGGTC TGAAAAAAGT 201 CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG 251 CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA 301 GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC 351 CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG 401 AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT 451 GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT 501 GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG 551 CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA 601 GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA 651 TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGAAAAAAGT ACCGACATCA 701 AAGCTGATAT CGCTACGAAc AAAGATAATA TTGCTAAAAA AGCAAACAGT 751 GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA 801 TGGTCTGAAC GCTACTACCG AAAAATTAGA CACACGCTTG GCTTCTGCTG 851 AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA 901 GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC 951 GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGAGGGGGTG 1001 GTGTCGCCGC CGACATCGGT GCGGGGCTTG CCGATGCACT AACCGCACCG 1051 CTCGACCATA AAGACAAAGG TTTGCAGTCT TTGACGCTGG ATCAGTCCGT 1101 CAGGAAAAAC GAGAAACTGA AGCTGGCGGC ACAAGGTGCG GAAAAAACTT 1151 ATGGAAACGG TGACAGCCTC AATACGGGCA AATTGAAGAA CGACAAGGTC 1201 AGCCGTTTCG ACTTTATCCG CCAAATCGAA GTGGACGGGC AGCTCATTAC 1251 CTTGGAGAGT GGAGAGTTCC AAGTATACAA ACAAAGCCAT TCCGCCTTAA 1301 CCGCCTTTCA GACCGAGCAA ATACAAGATT CGAAGCATTC CGGGAAGATG 1351 GTTGCGAAAC GCCAGTTCAG AATCGGCGAC ATAGCGGGCG AACATACATC 1401 TTTTGACAAG CTTCCCGAAG GTGGACGGGC GACATATCGC GGGACGGCGT 1451 TCGGTTCAGA CGATGCCGGC GGAAAACTGA CCTACACCAT AGATTTCGCC 1501 GCCAAGCAGG GAAACGGCAA AATCGAACAT TTGAAATCGC CAGAACTCAA 1551 TGTCGACCTG GCCGCCGCCG ATATCAAGCC GGATGGAAAA CGCCATGCCG 1601 TCATCAGCGG TTCCGTCCTT TACAACCAAG CCGAGAAAGG CAGTTACTCC 1651 CTCGGTATCT TTGGCGGAAA AGCCCAGGAA GTTGCCGGCA GCGCGGAAGT 1701 GAAAACCGTA AACGGCATAC GCCATATCGG CCTTGCCGCC AAGCAACTCG 1751 AGCACCACCA CCACCACCAC TGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL 101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 151 DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK 201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT 301 VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGVAADIG AGLADALTAP 351 LDHKDKGLQS LTLDQSVRKN EKLKLAAQGA EKTYGNGDSL NTGKLKNDKV 401 SRFDFIRQIE VDGQLITLES GEFQVYKQSH SALTAFQTEQ IQDSEHSGKM 451 VAKRQFRIGD IAGEHTSFDK LPEGGRATYR GTAFGSDDAG GKLTYTIDFA 501 AKQGNGKIEH LKSPELNVDL AAADIKPDGK RHAVISGSVL YNQAEKGSYS 551 LGIFGGKAQE VAGSAEVKTV NGIRHIGLAA KQLEHHHHHH * 961c-983 (SEQ ID NOS: 156 and 157) 1 ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC 51 TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA 101 CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT 151 GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT 201 CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG 251 CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA 301 GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC 351 CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG 401 AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT 451 GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT 501 GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG 551 CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA 601 GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA 651 TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA 701 AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT 751 GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA 801 TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG 851 AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA 901 GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC 951 GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGCGGAGGCG 1001 GCACTTCTGC GCCCGACTTC AATGCAGGCG GTACCGGTAT CGGCAGCAAC 1051 AGCAGAGCAA CAACAGCGAA ATCAGCAGCA GTATCTTACG CCGGTATCAA 1101 GAACGAAATG TGCAAAGACA GAAGCATGCT CTGTGCCGGT CGGGATGACG 1151 TTGCGGTTAC AGACAGGGAT GCCAAAATCA ATGCCCCCCC CCCGAATCTG 1201 CATACCGGAG ACTTTCCAAA CCCAAATGAC GCATACAAGA ATTTGATCAA 1251 CCTCAAACCT GCAATTGAAG CAGGCTATAC AGGACGCGGG GTAGAGGTAG 1301 GTATCGTCGA CACAGGCGAA TCCGTCGGCA GCATATCCTT TCCCGAACTG 1351 TATGGCAGAA AAGAACACGG CTATAACGAA AATTACAAAA ACTATACGGC 1401 GTATATGCGG AAGGAAGCGC CTGAAGACGG AGGCGGTAAA GACATTGAAG 1451 CTTCTTTCGA CGATGAGGCC GTTATAGAGA CTGAAGCAAA GCCGACGGAT 1501 ATCCGCCACG TAAAAGAAAT CGGACACATC GATTTGGTCT CCCATATTAT 1551 TGGCGGGCGT TCCGTGGACG GCAGACCTGC AGGCGGTATT GCGCCCGATG 1601 CGACGCTACA CATAATGAAT ACGAATGATG AAACCAAGAA CGAAATGATG 1651 GTTGCAGCCA TCCGCAATGC ATGGGTCAAG CTGGGCGAAC GTGGCGTGCG 1701 CATCGTCAAT AACAGTTTTG GAACAACATC GAGGGCAGGC ACTGCCGACC 1751 TTTTCCAAAT AGCCAATTCG GAGGAGCAGT ACCGCCAAGC GTTGCTCGAC 1801 TATTCCGGCG GTGATAAAAC AGACGAGGGT ATCCGCCTGA TGCAACAGAG 1851 CGATTACGGC AACCTGTCCT ACCACATCCG TAATAAAAAC ATGCTTTTCA 1901 TCTTTTCGAC AGGCAATGAC GCACAAGCTC AGCCCAACAC ATATGCCCTA 1951 TTGCCATTTT ATGAAAAAGA CGCTCAAAAA GGCATTATCA CAGTCGCAGG 2001 CGTAGACCGC AGTGGAGAAA AGTTCAAACG GGAAATGTAT GGAGAACCGG 2051 GTACAGAACC GCTTGAGTAT GGCTCCAACC ATTGCGGAAT TACTGCCATG 2101 TGGTGCCTGT CGGCACCCTA TGAAGCAAGC GTCCGTTTCA CCCGTACAAA 2151 CCCGATTCAA ATTGCCGGAA CATCCTTTTC CGCACCCATC GTAACCGGCA 2201 CGGCGGCTCT GCTGCTGCAG AAATACCCGT GGATGAGCAA CGACAACCTG 2251 CGTACCACGT TGCTGACGAC GGCTCAGGAC ATCGGTGCAG TCGGCGTGGA 2301 CAGCAAGTTC GGCTGGGGAC TGCTGGATGC GGGTAAGGCC ATGAACGGAC 2351 CCGCGTCCTT TCCGTTCGGC GACTTTACCG CCGATACGAA AGGTACATCC 2401 GATATTGCCT ACTCCTTCCG TAACGACATT TCAGGCACGG GCGGCCTGAT 2451 CAAAAAAGGC GGCAGCCAAC TGCAACTGCA CGGCAACAAC ACCTATACGG 2501 GCAAAACCAT TATCGAAGGC GGTTCGCTGG TGTTGTACGG CAACAACAAA 2551 TCGGATATGC GCGTCGAAAC CAAAGGTGCG CTGATTTATA ACGGGGCGGC 2601 ATCCGGCGGC AGCCTGAACA GCGACGGCAT TGTCTATCTG GCAGATACCG 2651 ACCAATCCGG CGCAAACGAA ACCGTACACA TCAAAGGCAG TCTGCAGCTG 2701 GACGGCAAAG GTACGCTGTA CACACGTTTG GGCAAACTGC TGAAAGTGGA 2751 CGGTACGGCG ATTATCGGCG GCAAGCTGTA CATGTCGGCA CGCGGCAAGG 2801 GGGCAGGCTA TCTCAACAGT ACCGGACGAC GTGTTCCCTT CCTGAGTGCC 2851 GCCAAAATCG GGCAGGATTA TTCTTTCTTC ACAAACATCG AAACCGACGG 2901 CGGCCTGCTG GCTTCCCTCG ACAGCGTCGA AAAAACAGCG GGCAGTGAAG 2951 GCGACACGCT GTCCTATTAT GTCCGTCGCG GCAATGCGGC ACGGACTGCT 3001 TCGGCAGCGG CACATTCCGC GCCCGCCGGT CTGAAACACG CCGTAGAACA 3051 GGGCGGCAGC AATCTGGAAA ACCTGATGGT CGAACTGGAT GCCTCCGAAT 3101 CATCCGCAAC ACCCGAGACG GTTGAAACTG CGGCAGCCGA CCGCACAGAT 3151 ATGCCGGGCA TCCGCCCCTA CGGCGCAACT TTCCGCGCAG CGGCAGCCGT 3201 ACAGCATGCG AATGCCGCCG ACGGTGTACG CATCTTCAAC AGTCTCGCCG 3251 CTACCGTCTA TGCCGACAGT ACCGCCGCCC ATGCCGATAT GCAGGGACGC 3301 CGCCTGAAAG CCGTATCGGA CGGGTTGGAC CACAACGGCA CGGGTCTGCG 3351 CGTCATCGCG CAAACCCAAC AGGACGGTGG AACGTGGGAA CAGGGCGGTG 3401 TTGAAGGCAA AATGCGCGGC AGTACCCAAA CCGTCGGCAT TGCCGCGAAA 3451 ACCGGCGAAA ATACGACAGC AGCCGCCACA CTGGGCATGG GACGCAGCAC 3501 ATGGAGCGAA AACAGTGCAA ATGCAAAAAC CGACAGCATT AGTCTGTTTG 3551 CAGGCATACG GCACGATGCG GGCGATATCG GCTATCTCAA AGGCCTGTTC 3601 TCCTACGGAC GCTACAAAAA CAGCATCAGC CGCAGCACCG GTGCGGACGA 3651 ACATGCGGAA GGCAGGATTA ACGGCACGCT GATGCAGCTG GGCGCACTGG 3701 GCGGTGTCAA CGTTCCGTTT GCCGCAACGG GAGATTTGAC GGTCGAAGGC 3751 GGTCTGCGCT ACGACCTGCT CAAACAGGAT GCATTCGCCG AAAAAGGCAG 3801 TGCTTTGGGC TGGAGCGGCA ACAGCCTCAC TGAAGGCACG CTGGTCGGAC 3851 TCGCGGGTCT GAAGCTGTCG CAACCCTTGA GCGATAAAGC CGTCCTGTTT 3901 GCAACGGCGG GCGTGGAACG CGACCTGAAC GGACGCGACT ACACGGTAAC 3951 GGGCGGCTTT ACCGGCGCGA CTGCAGCAAC CGGCAAGACG GGGGCACGCA 4001 ATATGCCGCA CACCCGTCTG GTTGCCGGCC TGGGCGCGGA TGTCGAATTC 4051 GGCAACGGCT GGAACGGCTT GGCACGTTAC AGCTACGCCG GTTCCAAACA 4101 GTACGGCAAC CACAGCGGAC GAGTCGGCGT AGGCTACCGG TTCCTCGAGC 4151 ACCACCACCA CCACCACTGA 1 MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT 51 AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL 101 ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA 151 DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK 201 AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIRADIATN KDNIAKKANS 251 ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT 301 VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGTSAPDF NAGGTGIGSN 351 SRATTAKSAA VSYAGIKNEM CKDRSMLCAG RDDVAVTDRD AKINAPPPNL 401 HTGDFPNPND AYKNLINLKP AIEAGYTGRG VEVGIVDTGE SVGSISFPEL 451 YGRKEHGYNE NYRNYTAYMR KEAPEDGGGK DIEASFDDEA VIETEAKPTD 501 IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDETKNEMM 551 VAAIRNAWVK LGERGVRIVN NSFGTTSRAG TADLFQIANS EEQYRQALLD 601 YSGGDKTDEG IRLMQQSDYG NLSYHIRNKN MLFIFSTGND AQAQPNTYAL 651 LPFYEKDAQK GIITVAGVDR SGEKFKREMY GEPGTEPLEY GSNHCGITAM 701 WCLSAFYEAS VRFTRTNPIQ IAGTSFSAPI VTGTAALLLQ KYPWMSNDNL 751 RTTLLTTAQD IGAVGVDSKF GWGLLDAGKA MNGPASFPFG DFTADTKGTS 801 DIAYSFRNDI SGTGGLIKKG GSQLQLHGNN TYTGKTIIEG GSLVLYGNNK 851 SDMRVETKGA LIYNGAASGG SLNSDGIVYL ADTDQSGANE TVHIKGSLQL 901 DGKGTLYTRL GKLLKVDGTA IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA 951 AKIGQDYSFF TNIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAARTA 1001 SAAAHSAPAG LRHAVEQGGS NLENLMVELD ASESSATPET VETAAADRTD 1051 MPGIRPYGAT FRAAAAVQHA NAADGVRIFN SLAATVYADS TAAHADMQGR 1101 RLKAVSDGLD HNGTGLRVIA QTQQDGGTWE QGGVEGKMRG STQTVGIAAK 1151 TGENTTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIRHDA GDIGYLKGLF 1201 SYGRYKNSIS RSTGADEHAE GSVNGTLMQL GALGGVNVPF AATGDLTVEG 1251 GLRYDLLKQD AFAEKGSALG WSGNSLTEGT LVGLAGLKLS QPLSDKAVLF 1301 ATAGVERDLN GRDYTVTGGF TGATAATGKT GARNMPHTRL VAGLGADVEF 1351 GNGWNGLARY SYAGSKQYGN HSGRVGVGYR FLEHHHHHH* 961cL-ORF46.1 (SEQ ID NOS: 158 and 159) 1 ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT 51 CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG 101 CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT 151 TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC 201 CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC 251 TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC 301 AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA 351 GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG 401 CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA 451 ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA 501 ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG 551 ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC 601 AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT 651 CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG 701 CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA 751 AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC 801 TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA 851 AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA 901 CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA 951 CGGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC 1001 TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT 1051 GGATCCGGAG GAGGAGGATC AGATTTGGCA AACGATTCTT TTATCCGGCA 1101 GGTTCTCGAC CGTCAGCATT TCGAACCCGA CGGGAAATAC CACCTATTCG 1151 GCAGCAGGGG GGAACTTGCC GAGCGCAGCG GCCATATCGG ATTGGGAAAA 1201 ATACAAASCC ATCAGTTGGG CAACCTGATG ATTCAACAGG CGGCCATTAA 1251 AGGAAATATC GGCTACATTG TCCGCTTTTC CGATCACGGG CACGAAGTCC 1301 ATTCCCCCTT CGACAACCAT GCCTCACATT CCGATTCTGA TGAAGCCGGT 1351 AGTCCCGTTG ACGGATTTAG CCTTTACCGC ATCCATTGGG ACGGATACGA 1401 ACACCATCCC GCCGACGGCT ATGACGGGCC ACAGGGCGGC GGCTATCCCG 1451 CTCCCAAAGG CGCGAGGGAT ATATACAGCT ACGACATAAA AGGCGTTGCC 1501 CAAAATATCC GCCTCAACCT GACCGACAAC CGCAGCACCG GACAACGGCT 1551 TGCCGACCGT TTCCACAATG CCGGTAGTAT GCTGACGCAA GGAGTAGGCG 1601 ACGGATTCAA ACGCGCCACC CGATACAGCC CCGAGCTGGA CAGATCGGGC 1651 AATGCCGCCG AAGCCTTCAA CGGCACTGCA GATATCGTTA AAAACATCAT 1701 CGGCGCGGCA GGAGAAATTG TCGGCGCAGG CGATGCCGTG CAGGGCATAA 1751 GCGAAGGCTC AAACATTGCT GTCATGCACG GCTTGGGTCT GCTTTCCACC 1801 GAAAACAAGA TGGCGCGCAT CAACGATTTG GCAGATATGG CGCAACTCAA 1851 AGACTATGCC GCAGCAGCCA TCCGCGATTG GGCAGTCCAA AACCCCAATG 1901 CCGCACAAGG CATAGAAGCC GTCAGCAATA TCTTTATGGC AGCCATCCCC 1951 ATCAAAGGGA TTGGAGCTGT TCGGGGAAAA TACGGCTTGG GCGGCATCAC 2001 GGCACATCCT ATCAAGCGGT CGCAGATGGG CGCGATCGCA TTGCCGAAAG 2051 GGAAATCCGC CGTCAGCGAC AATTTTGCCG ATGCGGCATA CGCCAAATAC 2101 CCGTCCCCTT ACCATTCCCG AAATATCCGT TCAAACTTGG AGCAGCGTTA 2151 CGGCAAAGAA AACATCACCT CCTCAACCGT GCCGCCGTCA AACGGCAAAA 2201 ATGTCAAACT GGCAGACCAA CGCCACCCGA AGACAGGCGT ACCGTTTGAC 2251 GGTAAAGGGT TTCCGAATTT TGAGAAGCAC GTGAAATATG ATACGTAACT 2301 CGAG 1 MKNFPSKVLT TAILATFCSG ALAATMDDDV KKAATVAIAA AYNNGQEING 51 FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN 101 KQNVDARVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI 151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV 201 KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA 251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSRFVRIDG LNATTERLDT 301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG 351 GSGGGGSDLA NDSFIRQVLD RQHFEPDGKY HLFGSRGELA ERSGHIGLGR 401 IQSHQLGNLM IQQAAIKGNI GYIVRFSDHG HEVHSPFDNH ASHSDSDEAG 451 SPVDGFSLYR IHWDGYEHHP ADGYDGAQGG GYPAPKGARD IYSYDIKGVA 501 QNIRLNLTDN RSTGQRLADR FHNAGSMLTQ GVGDGFKRAT RYSPELDRSG 551 NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV QGISEGSNIA VMHGLGLLST 601 ENKMARINDL ADMAQLKDYA AAAIRDWAVQ NPNAAQGIEA VSNIFMAAIP 651 IKGIGAVRGK YGLGGITAHP IKRSQMGAIA LPKGKSAVSD NFADAAYAKY 701 PSPYHSRNIR SNLEQRYGKE NITSSTVPPS NGKNVKLADQ RHPKTGVPFD 751 GKGFPNFEKH VRYDT* 961cL-741 (SEQ ID NOS: 160 and 161) 1 ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT 51 CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG 101 CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT 151 TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC 201 CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC 251 TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC 301 AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA 351 GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG 401 CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA 451 ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA 501 ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG 551 ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC 601 AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT 651 CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG 701 CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA 751 AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC 801 TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA 851 AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA 901 CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA 951 CGGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC 1001 TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT 1051 GGATCCGGAG GGGGTGGTGT CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA 1101 TGCACTAACC GCACCGCTCG ACCATAAAGA CAAAGGTTTG CAGTCTTTGA 1151 CGCTGGATCA GTCCGTCAGG AAAAACGAGA AACTGAAGCT GGCGGCACAA 1201 GGTGCGGAAA AAACTTATGG AAACGGTGAC AGCCTCAATA CGGGCAAATT 1251 GAAGAACGAC AAGGTCAGCC GTTTCGACTT TATCCGCCAA ATCGAAGTGG 1301 ACGGGCAGCT CATTACCTTG GAGAGTGGAG AGTTCCAAGT ATACAAACAA 1351 AGCCATTCCG CCTTAACCGC CTTTCAGACC GAGCAAATAC AAGATTCGGA 1401 GGATCCGGAG AAGATGGTTG CGAAACGCCA GTTCAGAATC GGCGACATAG 1451 CGGGCGAACA TACATCTTTT GACAAGCTTC CCGAAGGCGG CAGGGCGACA 1501 TATCGCGGGA CGGCGTTCGG TTCAGACGAT GCCGGCGGAA AACTGACCTA 1551 CACCATAGAT TTCGCCGCCA AGCAGGGAAA CGGCAAAATC GAACATTTGA 1601 AATCGCCAGA ACTCAATGTC GACCTGGCCG CCGCCGATAT CAAGCCGGAT 1651 GGAAAACGCC ATGCCGTCAT CAGCGGTTCC GTCCTTTACA ACCAAGCCGA 1701 GAAAGGCAGT TACTCCCTCG GTATCTTTGG CGGAAAAGCC CAGGAAGTTG 1751 CCGGCAGCGC GGAAGTGAAA ACCGTAAACG GCATACGCCA TATCGGCCTT 1801 GCCGCCAAGC AACTCGAGCA CCACCACCAC CACCACTGA 1 MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING 51 FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN 101 KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI 151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV 201 KTANEAKQTA EETKQNVDAK VKAAETAAGE AEAAAGTANT AADKAEAVAA 251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT 301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG 351 GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ 401 GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ 451 SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT 501 YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD 551 GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL 601 AAKQLEHHHH HH* 961cL-983 (SEQ ID NOS: 162 and 163) 1 ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT 51 CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG 101 CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT 151 TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC 201 CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC 251 TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC 301 AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA 351 GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG 401 CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA 451 ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA 501 ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG 551 ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC 601 AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT 651 CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG 701 CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA 751 AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC 801 TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA 851 AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA 901 CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA 951 CGGTTGGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC 1001 TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT 1051 GGATCCGGCG GAGGCGGCAC TTCTGCGCCC GACTTCAATG CAGGCGGTAC 1101 CGGTATCGGC AGCAACAGCA GAGCAACAAC AGCGAAATCA GCAGCAGTAT 1151 CTTACGCCGG TATCAAGAAC GAAATGTGCA AAGACAGAAG CATGCTCTGT 1201 GCCGGTCGGG ATGACGTTGC GGTTACAGAC AGGGATGCCA AAATCAATGC 1251 CCCCCCCCCG AATCTGCATA CCGGAGACTT TCCAAACCCA AATGACGCAT 1301 ACAAGAATTT GATCAACCTC AAACCTGCAA TTGAAGCAGG CTATACAGGA 1351 CGCGGGGTAG AGGTAGGTAT CGTCGACACA GGCGAATCCG TCGGCAGCAT 1401 ATCCTTTCCC GAACTGTATG GCAGAAAAGA ACACGGCTAT AACGAAAATT 1451 ACAAAAACTA TACGGCGTAT ATGCGGAAGG AAGCGCCTGA AGACGGAGGC 1501 GGTAAAGACA TTGAAGCTTC TTTCGACGAT GAGGCCGTTA TAGAGACTGA 1551 AGCAAAGCCG ACGGATATCC GCCACGTAAA AGAAATCGGA CACATCGATT 1601 TGGTCTCCCA TATTATTGGC GGGCGTTCCG TGGACGGCAG ACCTGCAGGC 1651 GGTATTGCGC CCGATGCGAC GCTACACATA ATGAATACGA ATGATGAAAC 1701 CAAGAACGAA ATGATGGTTG CAGCCATCCG CAATGCATGG GTCAAGCTGG 1751 GCGAACGTGG CGTGCGCATC GTCAATAACA GTTTIGGAAC AACATCGAGG 1801 GCAGGCACTG CCGACCTTTT CCAAATAGCC AATTCGGAGG AGCAGTACCG 1851 CCAAGCGTTG CTCGACTATT CCGGCGGTGA TAAAACAGAC GAGGGTATCC 1901 GCCTGATGCA ACAGAGCGAT TACGGCAACC TGTCCTACCA CATCCGTAAT 1951 AAAAACATGC TTTTCATCTT TTCGACAGGC AATGACGCAC AAGCTCAGCC 2001 CAACACATAT GCCCTATTGC CATTTTATGA AAAAGACGCT CAAAAAGGCA 2051 TTATCACAGT CGCAGGCGTA GACCGCAGTG GAGAAAAGTT CAAACGGGAA 2101 ATGTATGGAG AACCGGGTAC AGAACCGCTT GAGTATGGCT CCAACCATTG 2151 CGGAATTACT GCCATGTGGT GCCTGTCGGC ACCCTATGAA GCAAGCGTCC 2201 GTTTCACCCG TACAAACCCG ATTCAAATTG CCGGAACATC CTTTTCCGCA 2251 CCCATCGTAA CCGGCACGGC GGCTCTGCTG CTGCAGAAAT ACCCGTGGAT 2301 GAGCAACGAC AACCTGCGTA CCACGTTGCT GACGACGGCT CAGGACATCG 2351 GTGCAGTCGG CGTGGACAGC AAGTTCGGCT GGGGACTGCT GGATGCGGGT 2401 AAGGCCATGA ACGGACCCGC GTCCTTTCCG TTCGGCGACT TTACCGCCGA 2451 TACGAAAGGT ACATCCGATA TTGCCTACTC CTTCCGTAAC GACATTTCAG 2501 GCACGGGCGG CCTGATCAAA AAAGGCGGCA GCCAACTGCA ACTGCACGGC 2551 AACAACACCT ATACGGGCAA AACCATTATC GAAGGCGGTT CGCTGGTGTT 2601 GTACGGCAAC AACAAATCGG ATATGCGCGT CGAAACCAAA GGTGCGCTGA 2651 TTTATAACGG GGCGGCATCC GGCGGCAGCC TGAACAGCGA CGGCATTGTC 2701 TATCTGGCAG ATACCGACCA ATCCGGCGCA AACGAAACCG TACACATCAA 2751 AGGCAGTCTG CAGCTGGACG GCAAAGGTAC GCTGTACACA CGTTTGGGCA 2801 AACTGCTGAA AGTGGACGGT ACGGCGATTA TCGGCGGCAA GCTGTACATG 2851 TCGGCACGCG GCAAGGGGGC AGGCTATCTC AACAGTACCG GACGACGTGT 2901 TCCCTTCCTG AGTGCCGCCA AAATCGGGCA GGATTATTCT TTCTTCACAA 2951 ACATCGAAAC CGACGGCGGC CTGCTGGCTT CCCTCGACAG CGTCGAAAAA 3001 ACAGCGGGCA GTGAAGGCGA CACGCTGTCC TATTATGTCC GTCGCGGCAA 3051 TGCGGCACGG ACTGCTTCGG CAGCGGCACA TTCCGCGCCC GCCGGTCTGA 3101 AACACGCCGT AGAACAGGGC GGCAGCAATC TGGAAAACCT GATGGTCGAA 3151 CTGGATGCCT CCGAATCATC CGCAACACCC GAGACGGTTG AAACTGCGGC 3201 AGCCGACCGC ACAGATATGC CGGGCATCCG CCCCTACGGC GCAACTTTCC 3251 GCGCAGCGGC AGCCGTACAG CATGCGAATG CCGCCGACGG TGTACGCATC 3301 TTCAACAGTC TCGCCGCTAC CGTCTATGCC GACAGTACCG CCGCCCATGC 3351 CGATATGCAG GGACGCCGCC TGAAAGCCGT ATCGGACGGG TTGGACCACA 3401 ACGGCACGGG TCTGCGCGTC ATCGCGCAAA CCCAACAGGA CGGTGGAACG 3451 TGGGAACAGG GCGGTGTTGA AGGCAAAATG CGCGGCAGTA CCCAAACCGT 3501 CGGCATTGCC GCGAAAACCG GCGAAAATAC GACAGCAGCC GCCACACTGG 3551 GCATGGGACG CAGCACATGG AGCGAAAACA GTGCAAATGC AAAAACCGAC 3601 AGCATTAGTC TGPPTGCAGG CATACGGCAC GATGCGGGCG ATATCGGCTA 3651 TCTCAAAGGC CTGTTCTCCT ACGGACGCTA CAAAAACAGC ATCAGCCGCA 3701 GCACCGGTGC GGACGAACAT GCGGAAGGCA GCGTCAACGG CACGCTGATG 3751 CAGCTGGGCG CACTGGGCGG TGTCAACGTT CCGTPPGCCG CAACGGGAGA 3801 TTTGACGGTC GAAGGCGGTC TGCGCTACGA CCTGCTCAAA CAGGATGCAT 3851 TCGCCGAAAA AGGCAGTGCT TTGGGCTGGA GCGGCAACAG CCTCACTGAA 3901 GGCACGCTGG TCGGACTCGC GGGTCTGAAG CTUTCGCAAC CCTTGAGCGA 3951 TAAAGCCGTC CTGTTTGCAA CGGCGGGCGT GGAACGCGAC CTGAACGGAC 4001 GCGACTACAC GGTAACGGGC GGCTTTACCG GCGCGACTGC AGCAACCGGC 4051 AAGACGGGGG CACGCAATAT GCCGCACACC CGTCTGGTTG CCGGCCTGGG 4101 CGCGGATGTC GAATTCGGCA ACGGCTGGAA CGGCTTGGCA CGTTACAGCT 4151 ACGCCGGTTC CAAACAGTAC GGCAACCACA GCGGACGAGT CGGCGTAGGC 4201 TACCGGTTCT GACTCGAG 1 MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNEGQEING 51 FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN 101 KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI 151 TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV 201 KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA 251 KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT 301 RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG 351 GSGGGGTSAP DFNAGGTGIG SNSRATTAKS AAVSYAGIKN EMCKDRSMLC 401 AGRDDVAVTD RDAKINAPPP NLHTGDFPNP NDAYKNLINL KPAIEAGYTG 451 RGVEVGIVDT GESVGSISFP ELYGRKEHGY NENYKNYTAY MRKEAPEDGG 501 GKDIEASFDD EAVIETEAKP TDIRHVKEIG HIDLVSHIIG GRSVDGRPAG 551 GIAPDATLHI MNTNDETKNE MMVAAIRNAW VKLGERGVRI VNNSFGTTSR 601 AGTADLFQIA NSEEQYRQAL LDYSGGDKTD EGIRLMQQSD YGNLSYHIRN 651 KNMLFIFSTG NDAQAQPNTY ALLPFYEKDA QKGIITVAGV DRSGEKFKRE 701 MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVRFTRTNP IQIAGTSFSA 751 PIVTGTAALL LQKYPWMSND NLRTTLLTTA QDIGAVGVDS KFGWGLLDAG 801 KAMNGPASFP FGDFTADTKG TSDIAYSFRN DISGTGGLIK KGGSQLQLHG 851 NNTYTGRTII EGGSLVLYGN NKSDMRVETK GALIYNGAAS GGSLNSDGIV 901 YLADTDQSGA NETVHIKGSL QLDGKGTLYT RLGKLLKVDG TAIIGGKLYM 951 SARGRGAGYL NSTGRRVPFL SAAKIGQDYS FFTNIETDGG LLASLDSVEK 1001 TAGSEGDTLS YYVRRGNAAR TASAAAHSAP AGLKHAVEQG GSNLENLMVE 1051 LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVQ HANAADGVRI 1101 FNSLAATVYA DSTAAHADMQ GRRLKAVSDG LDHNGTGLRV IAQTQQDGGT 1151 WEQGGVEGKM RGSTQTVGIA AKTGENTTAA ATLGMGRSTW SENSANAKTD 1201 SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTLM 1251 QLGALGGVNV PFAATGDLTV EGGLRYDLLK QDAFAEKGSA LGWSGNSLTE 1301 GTLVGLAGLK LSQPLSDKAV LFATAGVERD LNGRDYTVTG GFTGATAATG 1351 KTGARNMPHT RLVAGLGADV EFGNGWNGLA RYSYAGSKQY GNHSGRVGVG 1401 YRF*

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. For instance, the use of proteins from other strains is envisaged [e.g. see WO00/66741 for polymorphic sequences for ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953].

EXPERIMENTAL DETAILS FPLC Protein Purification

The following table summarises the FPLC protein purification that was used:

Protein PI Column Buffer pH Protocol 121.1^(untagged) 623 Mono Q Tris 8.0 A 128.1^(untagged) 5.04 Mono Q Bis-Tris propane 6.5 A 406.1L 7.75 Mono Q Diethanolamine 9.0 B 576.1L 5.63 Mono Q Tris 7.5 B 593^(untagged) 8.79 Mono S Hepes 7.4 A 726^(untagged) 4.95 Hi-trap S Bis-Tris 6.0 A 919^(untagged) 10.5(-leader) Mono S Bicine 8.5 C 919Lorf4 10.4(-leader) Mono S Tris 8.0 B 920L 6.92(-leader) Mono Q Diethanolamine 8.5 A 953L 7.56(-leader) Mono S MES 6.6 D 982^(untagged) 4.73 Mono Q Bis-Tris propane 6.5 A 919-287 6.58 Hi-trap Q Tris 8.0 A 953-287 4.92 Mono Q Bis-Tris propane 6.2 A

Buffer solutions included 20-120 mM NaCl, 5.0 mg/ml CHAPS and 10% v/v glycerol. The dialysate was centrifuged at 13000 g for 20 min and applied to either a mono Q or mono S FPLC ion-exchange resin. Buffer and ion exchange resins were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual [Pharmacia: FPLC Ion Exchange and Chromatofocussing; Principles and Methods. Pharmacia Publication]. Proteins were eluted using a step-wise NaCl gradient. Purification was analysed by SDS-PAGE and protein concentration determined by the Bradford method.

The letter in the ‘protocol’ column refers to the following:

FPLC-A: Clones 121.1, 128.1, 593, 726, 982, periplasmic protein 920L and hybrid proteins 919-287, 953-287 were purified from the soluble fraction of E. coli obtained after disruption of the cells. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at either 30° C. or 37° C. until the OD₅₅₀ reached 0.6-08. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed on ice or at 4° C. For cytosolic proteins (121.1, 128.1, 593, 726 and 982) and periplasmic protein 920L, bacteria were resuspended in 25 ml of PBS containing complete protease inhibitor (Boehringer-Mannheim). Cells were lysed by sonication using a Branson SONIFIER® 450 (ultrasonic cell disruption/homogenizer). Disrupted cells were centrifuged at 8000 g for 30 min to sediment unbroken cells and inclusion bodies and the supernatant taken to 35% v/v saturation by the addition of 3.9 M (NH₄)₂SO₄. The precipitate was sedimented at 8000 g for 30 minutes. The supernatant was taken to 70% v/v saturation by the addition of 3.9 M (NH₄)₂SO₄ and the precipitate collected as above. Pellets containing the protein of interest were identified by SDS-PAGE and dialysed against the appropriate ion-exchange buffer (see below) for 6 hours or overnight. The periplasmic fraction from E. coli expressing 953L was prepared according to the protocol of Evans et. al. [Infect. Immun. (1974) 10:1010-1017] and dialysed against the appropriate ion-exchange buffer. Buffer and ion exchange resin were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual (Pharmacia). Buffer solutions included 20 mM NaCl, and 10% (v/v) glycerol. The dialysate was centrifuged at 13000 g for 20 min and applied to either a mono Q or mono S FPLC ion-exchange resin. Buffer and ion exchange resin were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual (Pharmacia). Proteins were eluted from the ion-exchange resin using either step-wise or continuous NaCl gradients. Purification was analysed by SDS-PAGE and protein concentration determined by Bradford method. Cleavage of the leader peptide of periplasmic proteins was demonstrated by sequencing the NH₂-terminus (see below).

FPLC-B: These proteins were purified from the membrane fraction of E. coli. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium. Clones 406.1L and 919LOrf4 were grown at 30° C. and Orf25L and 576.1L at 37° C. until the OD₅₅₀ reached 0.6-0.8. In the case of 919LOrf4, growth at 30° C. was essential since expression of recombinant protein at 37° C. resulted in lysis of the cells. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed at 4° C. Bacteria were resuspended in 25 ml of PBS containing complete protease inhibitor (Boehringer-Mannheim) and lysed by osmotic shock with 2-3 passages through a French Press. Unbroken cells were removed by centrifugation at 5000 g for 15 min and membranes precipitated by centrifugation at 100000 g (Beckman Ti50, 38000 rpm) for 45 minutes. A Dounce homogenizer was used to re-suspend the membrane pellet in 7.5 ml of 20 mM Tris-HCl (pH 8.0), 1.0 M NaCl and complete protease inhibitor. The suspension was mixed for 2-4 hours, centrifuged at 100000 g for 45 min and the pellet resuspended in 7.5 ml of 20 mM Tris-HCl (pH 8.0), 1.0M NaCl, 5.0 mg/ml CHAPS, 10% (v/v) glycerol and complete protease inhibitor. The solution was mixed overnight, centrifuged at 100000 g for 45 minutes and the supernatant dialysed for 6 hours against an appropriately selected buffer. In the case of Orf25.L, the pellet obtained after CHAPS extraction was found to contain the recombinant protein. This fraction, without further purification, was used to immunise mice.

FPLC-C: Identical to FPLC-A, but purification was from the soluble fraction obtained after permeabilising E. coli with polymyxin B, rather than after cell disruption.

FPLC-D: A single colony harbouring the plasmid of interest was grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at 30° C. until the OD₅₅₀ reached 0.6-0.8. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed on ice or at 4° C. Cells were resuspended in 20 mM Bicine (pH 8.5), 20 mM NaCl, 10% (v/v) glycerol, complete protease inhibitor (Boehringer-Mannheim) and disrupted using a Branson SONIFIER® 450 (ultrasonic cell disruption/homogenizer). The sonicate was centrifuged at 8000 g for 30 min to sediment unbroken cells and inclusion bodies. The recombinant protein was precipitated from solution between 35% v/v and 70% v/v saturation by the addition of 3.9M (NH₄)₂SO₄. The precipitate was sedimented at 8000 g for 30 minutes, resuspended in 20 mM Bicine (pH 8.5), 20 mM NaCl, 10% (v/v) glycerol and dialysed against this buffer for 6 hours or overnight. The dialysate was centrifuged at 13000 g for 20 min and applied to the FPLC resin. The protein was eluted from the column using a step-wise NaCl gradients. Purification was analysed by SDS-PAGE and protein concentration determined by Bradford method.

Cloning Strategy and Oligonucleotide Design

Genes coding for antigens of interest were amplified by PCR, using oligonucleotides designed on the basis of the genomic sequence of N. meningitidis B MC58. Genomic DNA from strain 2996 was always used as a template in PCR reactions, unless otherwise specified, and the amplified fragments were cloned in the expression vector pET21b+(Novagen) to express the protein as C-terminal His-tagged product, or in pET-24b+(Novagen) to express the protein in ‘untagged’ form (e.g. ΔG 287K).

Where a protein was expressed without a fusion partner and with its own leader peptide (if present), amplification of the open reading frame (ATG to STOP codons) was performed.

Where a protein was expressed in ‘untagged’ form, the leader peptide was omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence.

The melting temperature of the primers used in PCR depended on the number and type of hybridising nucleotides in the whole primer, and was determined using the formulae: T _(m1)=4(G+C)+2(A+T)  (tail excluded) T _(m2)=64.9+0.41(% GC)−600/N   (whole primer)

The melting temperatures of the selected oligonucleotides were usually 65-70° C. for the whole oligo and 50-60° C. for the hybridising region alone.

Oligonucleotides were synthesised using a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2.0 ml NH₄OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were centrifuged and the pellets resuspended in water.

Restriction Sequences SEQ ID NO site Orf1L Fwd CGCGGATCCGCTAGC-AAAACAACCGACAAACGG 164 NheI Rev CCCGCTCGAG-TTACCAGCGGTAGCCTA 165 XhoI Orf1 Fwd CTAGCTAGC-GGACACACTTATTTCGGCATC 166 NheI Rev CCCGCTCGAG-TTACCAGCGGTAGCCTAATTTG 167 XhoI Orf1LOmpA Fwd NdeI-(NheI) Rev CCCGCTCGAG- 168 XhoI Orf4L Fwd CGCGGATCCCATATG-AAAACCTTCTTCAAAACC 169 NdeI Rev CCCGCTCGAG-TTATTTGGCTGCGCCTTC 170 XhoI Orf7-1L Fwd GCGGCATTAAT-ATGTTGAGAAAATTGTTGAAATGG 171 AseI Rev GCGGCCTCGAG-TTATTTTTTCAAAATATATTGC 172 XhoI Orf9-1L Fwd GCGGCCATATG-TTACCTAACCGTTTCAAAATGT 173 NdeI Rev GCGGCCTCGAG-TTATTTCCGAGGTTTTCGGG 174 XhoI Orf23L Fwd CGCGGATCCCATATG-ACACGCTTCAAATATTC 175 NdeI Rev CCCGCTCGAG-TTATTTAAACCGATAGGTAAA 176 XhoI Orf25-1 His Fwd CGCGGATCCCATATG-GGCAGGGAAGAACCGC 177 NdeI Rev GCCCAAGCTT-ATCGATGGAATAGCCGCG 178 HindIII Orf29-1 b-His Fwd CGCGGATCCGCTAGC-AACGGTTTGGATGCCCG 179 NheI (MC58) Rev CCCGCTCGAG-TTTGTCTAAGTTCCTGATAT 180 XhoI CCCGCTCGAG-ATTCCCACCTGCCATC 181 Orf29-1 b-L Fwd CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT 182 NheI (MC58) Rev CCCGCTCGAG-TTAATTCCCACCTGCCATC 183 XhoI Orf29-1 c-His Fwd CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT 184 NheI (MC58) Rev CCCGCTCGAG-TTGGACGATGCCCGCGA 185 XhoI Orf29-1 c-L Fwd CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT 186 NheI (MC58) Rev CCCGCTCGAG-TTATTGGACGATGCCCGC 187 XhoI Orf25L Fwd CGCGGATCCCATATG-TATCGCAAACTGATTGC 188 NdeI Rev CCCGCTCGAG-CTAATCGATGGAATAGCC 189 XhoI Orf37L Fwd CGCGGATCCCATATG-AAACAGACAGTCAAATG 190 NdeI Rev CCCGCTCGAG-TCAATAACCCGCCTTCAG 191 XhoI Orf38L Fwd CGCGGATCCCATATG-TTACGTTTGACTGCTTTAGCCGTATGCACC 192 NdeI Rev CCCGCTCGAG-TTATTTTGCCGCGTTAAAAGCGTCGGCAAC 193 XhoI Orf40L Fwd CGCGGATCCCATATG-AACAAAATATACCGCAT 194 NdeI Rev CCCGCTCGAG-TTACCACTGATAACCGAC 195 XhoI Orf40.2-His Fwd CGCGGATCCCATATG-ACCGATGACGACGATTTAT 196 NdeI Rev GCCCAAGCTT-CCACTGATAACCGACAGA 197 HindIII Orf40.2L Fwd CGCGGATCCCATATG-AACAAAATATACCGCAT 198 NdeI Rev GCCCAAGCTT-TTACCACTGATAACCGAC 199 HindIII Orf46-2L Fwd GGGAATTCCATATG-GGCATTTCCCGCAAAATATC 200 NdeI Rev CCCGCTCGAG-TTATTTACTCCTATAACGAGGTCTCTTAAC 201 XhoI Orf46-2 Fwd GGGAATTCCATATG-TCAGATTTGGCAAACGATTCTT 202 NdeI Rev CCCGCTCGAG-TTATTTACTCCTATAACGAGGTCTCTTAAC 203 XhoI Orf46.1L Fwd GGGAATTCCATATG-GGCATTTCCCGCAAAATATC 204 NdeI Rev CCCGCTCGAG-TTACGTATCATATTTCACGTGC 205 XhoI orf46. (His-GST) Fwd GGGAATTCCATATGCACGTGAAATATGATACGAAG 206 BamHI-NdeI Rev CCCGCTCGAGTTTACTCCTATAACGAGGTCTCTTAAC 207 XhoI rf46.1-His Fwd GGGAATTCCATATGTCAGATTTGGCAAACGATTCTT 208 NdeI Rev CCCGCTCGAGCGTATCATATTTCACGTGC 209 XhoI orf46.2-His Fwd GGGAATTCCATATGTCAGATTTGGCAAACGATTCTT 210 NdeI Rev CCCGCTCGAGTTTACTCCTATAACGAGGTCTCTTAAC 211 XhoI Orf65-1-(His/GST) Fwd CGCGGATCCCATATG-CAAAATGCGTTCAAAATCCC 212 BamHI-NdeI (MC58) Rev CGCGGATCCCATATG-AACAAAATATACCGCAT 213 XhoI CCCGCTCGAG-TTTGCTTTCGATAGAACGG 214 Orf72-1L Fwd GCGGCCATATG-GTCATAAAATATACAAATTTGAA 215 NdeI Rev GCGGCCTCGAG-TTAGCCTGAGACCTTTGCAAATT 216 XhoI Orf76-1L Fwd GCGGCCATATG-AAACAGAAAAAAACCGCTG 217 NdeI Rev GCGGCCTCGAG-TTACGGTTTGACACCGTTTTC 218 XhoI Orf83.1L Fwd CGCGGATCCCATATG-AAAACCCTGCTCCTC 219 NdeI Rev CCCGCTCGAG-TTATCCTCCTTTGCGGC 220 XhoI Orf85-2L Fwd GCGGCCATATG-GCAAAAATGATGAAATGGG 221 NdeI Rev GCGGCCTCGAG-TTATCGGCGCGGCGGGCC 222 Xhol Orf91L (MC58) Fwd GCGGCCATATGAAAAAATCCTCCCTCATCA 223 NdeI Rev GCGGCCTCGAGTTATTTGCCGCCGTTTTTGGC 224 XhoI Orf91-His (MC58) Fwd GCGGCCATATGGCCCCTGCCGACGCGGTAAG 225 NdeI Rev GCGGCCTCGAGTTTGCCGCCGTTTTTGGCTTTC 226 XhoI Orf97-1L Fwd GCGGCCATATG-AAACACATACTCCCCCTGA 227 NdeI Rev GCGGCCTCGAG-TTATTCGCCTACGGTTTTTTG 228 XhoI Orf119L (MC58) Fwd GCGGCCATATGATTTACATCGTACTGTTTC 229 NdeI Rev GCGGCCTCGAGTTAGGAGAACAGGCGCAATGC 230 XhoI Orf119-His (MC58) Fwd GCGGCCATATGTACAACATGTATCAGGAAAAC 231 NdeI Rev GCGGCCTCGAGGGAGAACAGGCGCAATGCGG 232 XhoI Orf137.1 (His-GST) Fwd CGCGGATCCGCTAGCTGCGGCACGGCGGG 233 BamHI-NheI (MC58) Rec CCCGCTCGAGATAACGGTATGCCGCCAG 234 XhoI Orf143-1L Fwd CGCGGATCCCATATG-GAATCAACACTTTCAC 235 NdeI Rev CCCGCTCGAG-TTACACGCGGTTGCTGT 236 XhoI 008 Fwd CGCGGATCCCATATG-AACAACAGACATTTTG 237 NdeI Rev CCCGCTCGAG-TTACCTGTCCGGTAAAAG 238 XhoI 050-1 (48) Fwd CGCGGATCCGCTAGC-ACCGTCATCAAACAGGAA 239 NheI Rev CCCGCTCGAG-TCAAGATTCGACGGGGA 240 XhoI 105 Fwd CGCGGATCCCATATG-TCCGCAAACGAATACG 241 NdeI Rev CCCGCTCGAG-TCAGTGTTCTGCCAGTTT 242 XhoI 111L Fwd CGCGGATCCCATATG-CCGTCTGAAACACG 243 Ndel Rev CCCGCTCGAG-TTAGCGGAGCAGTTTTTC 244 XhoI 117-1 Fwd CGCGGATCCCATATG-ACCGCCATCAGCC 245 NdeI Rev CCCGCTCGAG-TTAAAGCCGGGTAACGC 246 XhoI 121-1 Fwd GCGGCCATATG-GAAACACAGCTTTACATCGG 247 NdeI Rev GCGGCCTCGAG-TCAATAATAATATCCCGCG 248 XhoI 122-1 Fwd GCGGCCATATG-ATTAAAATCCGCAATATCC 249 NdeI Rev GCGGCCTCGAG-TTAAATCTTGGTAGATTGGATTTGG 250 XhoI 128-1 Fwd GCGGCCATATG-ACTGACAACGCACTGCTCC 251 NdeI Rev GCGGCCTCGAG-TCAGACCGCGTTGTCGAAAC 252 XhoI 148 Fwd CGCGGATCCCATATG-GCGTTAAAAACATCAAA 253 NdeI Rev CCCGCTCGAG-TCAGCCCTTCATACAGC 254 XhoI 149.1L (MC58) Fwd GCGGCATTAATGGCACAAACTACACTCAAACC 255 AseI Rev GCGGCCTCGAGTTAAAACTTCACGTTCACGCCG 256 XhoI 149.1-His (MC58) Fwd GCGGCATTAATGCATGAAACTGAGCAATCGGTGG 257 AseI Rev GCGGCCTCGAGAAACTTCACGTTCACGCCGCCGGTAAA 258 XhoI 205 (His-GST) Fwd CGCGGATCCCATATGGGCAAATCCGAAAATACG 259 BamHI-NdeI (MC58) Rev CCCGCTCGAGATAATGGCGGCGGCGG 260 XhoI 206L Fwd CGCGGATCCCATATG-TTTCCCCCCGACAA 261 NdeI Rev CCCGCTCGAG-TCATTCTGTAAAAAAAGTATG 262 XhoI 214 (His-GST) Fwd CGCGGATCCCATATGCTTCAAAGCGACAGCAG 263 BamHI-NdeI (MC58) Rev CCCGCTCGAGTTCGGATTTTTGCGTACTC 264 XhoI 216 Fwd CGCGGATCCCATATG-GCAATGGCAGAAAACG 265 NdeI Rev CCCGCTCGAG-CTATACAATCCGTGCCG 266 XhoI 225-1L Fwd CGCGGATCCCATATG-GATTCTTTTTTCAAACC 267 NdeI Rev CCCGCTCGAG-TCAGTTCAGAAAGCGGG 268 Xhol 235L Fwd CGCGGATCCCATATG-AAACCTTTGATTTTAGG 269 NdeI Rev CCCGCTCGAG-TTATTTGGGCTGCTCTTC 270 Xhol 243 Fwd CGCGGATCCCATATG-GTAATCGTCTGGTTG 271 NdeI Rev CCCGCTCGAG-CTACGACTTGGTTACCG 272 XhoI 247-1L Fwd GCGGCCATATG-AGACGTAAAATGCTAAAGCTAC 273 NdeI Rev GCGGCCTCGAG-TCAAAGTGTTCTGTTTGCGC 274 XhoI 264-His Fwd GCCGCCATATG-TTGACTTTAACCCGAAAAA 275 NdeI Rev GCCGCCTCGAG-GCCGGCGGTCAATACCGCCCGAA 276 XhoI 270 (His-GST) Fwd CGCGGATCCCATATGGCGCAATGCGATTTGAC 277 BamHI-NdeI (MC58) Rev CCCGCTCGAGTTCGGCGGTAAATGCCG 278 XhoI 274L Fwd GCGGCCATATG-GCGGGGCCGATTTTTGT 279 NdeI Rev GCGGCCTCGAG-TTATTTGCTTTCAGTATTATTG 280 Xhol 283L Fwd GCGGCCATATG-AACTTTGCTTTATCCGTCA 281 NdeI Rev GCGGCCTCGAG-TTAACGGCAGTATTTGTTTAC 282 XhoI 285-His Fwd CGCGGATCCCATATGGGTTTGCGCTTCGGGC 283 BamHI Rev GCCCAAGCTTTTTTCCTTTGCCGTTTCCG 284 HindIII 286-His Fwd CGCGGATCCCATATG-GCCGACCTTTCCGAAAA 285 NdeI (MC58) Rev CCCGCTCGAG-GAAGCGCGTTCCCAAGC 286 XhoI 286L Fwd CGCGGATCCCATATG-CACGACACCCGTAC 287 NdeI (MC58) Rev CCCGCTCGAG-TTAGAAGCGCGTTCCCAA 288 XhoI 287L Fwd CTAGCTAGC-TTTAAACGCAGCGTAATCGCAATGG 289 NheI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 290 XhoI 287 Fwd CTAGCTAGC-GGGGGCGGCGGTGGCG 291 NheI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 292 XhoI 287LOrf4 Fwd CTAGCTAGCGCTCATCCTCGCCGCC-TGCGGGGGCGGCGGT 293 NheI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 294 XhoI 287-fu Fwd CGGGGATCC-GGGGGCGGCGGTGGCG 295 BamHI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTTGCC 296 XhoI 287-His Fwd CTAGCTAGC-GGGGGCGGCGGTGGCG 297 NheI Rev CCCGCTCGAG-ATCCTGCTCTTTTTTGCC * 298 XhoI 287-His (2996) Fwd CTAGCTAGC-TGCGCTGGGCGGCGGTGGCG 299 NheI Rev CCCGCTCGAG-ATCCTGCTCTTTTTTGCC 300 XhoI Δ1 287-His Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC ^(§) 301 NheI Δ2 287-His Fwd CGCGGATCCGCTAGC-CAAGATATGGCGGCAGT ^(§) 302 NheI Δ3 287-His Fwd CGCGGATCCGCTAGC-GCCGAATCCGCAAATCA ^(§) 303 NheI Δ4 287-His Fwd CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG ^(§) 304 NheI Δ4 287MC58-His Fwd CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG ^(§) 305 NheI 287a-His Fwd CGCCATATG-TTTAAACGCAGCGTAATCGC 306 NdeI Rev CCCGCTCGAG-AAAATTGCTACCGCCATTCGCAGG 307 XhoI 287b-His Fwd CGCCATATG-GGAAGGGTTGATTTGGCTAATGG 308 NdeI 287b-2996-His Rev CCCGCTCGAG-CTTGTCTTTATAAATGATGACATATTTG 309 XhoI 287b-MC58-His Rev CCCGCTCGAG-TTTATAAAAGATAATATATTGATTGATTCC 310 XhoI 287c-2996-His Fwd CGCGCTAGC-ATGCCGCTGATTCCCGTCAATC ^(§) 311 NheI ‘287^(untagged)’ (2996) Fwd CTAGCTAGC-GGGGGCGGCGGTGGCG 312 NheI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 313 XhoI ΔG287-His * Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC 314 NheI Rev CCCGCTCGAG-ATCCTGCTCTTTTTTGCC 315 XhoI ΔG287K (2996) Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC 316 NheI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 317 XhoI ΔG 287-L Fwd CGCGGATCCGCTAGC- 318 NheI TTTGAACGCAGTGTGATTGCAATGGCTTGTATTTTTGCC CTTTCAGCCTGT TCGCCCGATGTTAAATCGGCG Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 319 XhoI ΔG 287-Orf4L Fwd CGCGGATCCGCTAGC- 320 NheI AAAACCTTCTTCAAAACCCTTTCCGCCGCCGCACTCGCG CTCATCCTCGCCGCCTGC TCGCCCGATGTTAAATCG Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 321 XhoI 292L Fwd CGCGGATCCCATATG-AAAACCAAGTTAATCAAA 322 NdeI Rev CCCGCTCGAG-TTATTGATTTTTGCGGATGA 323 XhoI 308-1 Fwd CGCGGATCCCATATG-TTAAATCGGGTATTTTATC 324 NdeI Rev CCCGCTCGAG-TTAATCCGCCATTCCCTG 325 XhoI 401L Fwd GCGGCCATATG-AAATTACAACAATTGGCTG 326 NdeI Rev GCGGCCTCGAG-TTACCTTACGTTTTTCAAAG 327 XhoI 406L Fwd CGCGGATCCCATATG-CAAGCACGGCTGCT 328 NdeI Rev CCCGCTCGAG-TCAAGGTTGTCCTTGTCTA 329 XhoI 502-1L Fwd CGCGGATCCCATATG-ATGAAACCGCACAAC 330 NdeI Rev CCCGCTCGAG-TCAGTTGCTCAACACGTC 331 XhoI 502-A (His-GST) Fwd CGCGGATCCCATATGGTAGACGCGCTTAAGCA 332 BamHI-NdeI Rev CCCGCTCGAGAGCTGCATGGCGGCG 333 XhoI 503-1L Fwd CGCGGATCCCATATG-GCACGGTCGTTATAC 334 NdeI Rev CCCGCTCGAG-CTACCGCGCATTCCTG 335 XhoI 519-1L Fwd GCGGCCATATG-GAATTTTTCATTATCTTGTT 336 NdeI Rev GCGGCCTCGAG-TTATTTGGCGGTTTTGCTGC 337 XhoI 525-1L Fwd GCGGCCATATG-AAGTATGTCCGGTTATTTTTC 338 NdeI Rev GCGGCCTCGAG-TTATCGGCTTGTGCAACGG 339 XhoI 529-(His/GST) Fwd CGCGGATCCGCTAGC-TCCGGCAGCAAAACCGA 340 Bam HI-NheI (MC58) Rev GCCCAAGCTT-ACGCAGTTCGGAATGGAG 341 HindIII 552L Fwd GCCGCCATATGTTGAATATTAAACTGAAAACCTTG 342 NdeI Rev GCCGCCTCGAGTTATTTCTGATGCCTTTTCCC 343 XhoI 556L Fwd GCCGCCATATGGACAATAAGACCAAACTG 344 NdeI Rev GCCGCCTCGAGTTAACGGTGCGGACGTTTC 345 XhoI 557L Fwd CGCGGATCCCATATG-AACAAACTGTTTCTTAC 346 NdeI Rev CCCGCTCGAG-TCATTCCGCCTTCAGAAA 347 XhoI 564ab-(His/GST) Fwd CGCGGATCCCATATG-CAAGGTATCGTTGCCGACAAATCCGCACCT 348 BamHI-NdeI (MC58) Rev CCCGCTCGAG-AGCTAATTGTGCTTGGTTTGCAGATAGGAGTT 349 XhoI 564abL (MC58) Fwd CGCGGATCCCATATG-AACCGCACCCTGTACAAAGTTGTATTTAACAAACATC 350 NdeI Rev CCCGCTCGAG-TTAAGCTAATTGTGCTTGGTTTGCAGATAGGAGTT 351 XhoI 564b-(His/GST) Fwd CGCGGATCCCATATG-ACGGGAGAAAATCATGCGGTTTCACTTCATG 352 BamHI-NdeI (MC58) Rev CCCGCTCGAG-AGCTAATTGTGCTTGGTTTGCAGATAGGAGTT 353 XhoI 564c-(His/GST) Fwd CGCGGATCCCATATG-GTTTCAGACGGCCTATACAACCAACATGGTGAAATT 354 BamHI-NdeI (MC58) Rev CCCGCTCGAG-GCGGTAACTGCCGCTTGCACTGAATCCGTAA 355 XhoI 564bc-(His/GST) Fwd CGCGGATCCCATATG-ACGGGAGAAAATCATGCGGTTTCACTTCATG 356 BamHI-NdeI (MC58) Rev CCCGCTCGAG-GCGGTAACTGCCGCTTGCACTGAATCCGTAA 357 XhoI 564d-(His/GST) Fwd CGCGGATCCCATATG-CAAAGCAAAGTCAAAGCAGACCATGCCTCCGTAA 358 BamHI-NdeI (MC58) Rev CCCGCTCGAG-TCTTTTCCTTTCAATTATAACTTTAGTAGGTTCAATTTTG 359 XhoI GTCCCC 564cd- Fwd CGCGGATCCCATATG-GTTTCAGACGGCCTATACAACCAACATGGTGAAATT 360 BamHI-NdeI (His/GST)(MC58) Rev CCCGCTCGAG-TCTTTTCCTTTCAATTATAACTTTAGTAGGTTCAATTTTG 361 XhoI GTCCCC 570L Fwd GCGGCCATATG-ACCCGTTTGACCCGCG 362 NdeI Rev GCGGCCTCGAG-TCAGCGGGCGTTCATTTCTT 363 XhoI 576-1L Fwd CGCGGATCCCATATG-AACACCATTTTCAAAATC 364 NdeI Rev CCCGCTCGAG-TTAATTTACTTTTTTGATGTCG 365 XhoI 580L Fwd GCGGCCATATG-GATTCGCCCAAGGTCGG 366 NdeI Rev GCGGCCTCGAG-CTACACTTCCCCCGAAGTGG 367 XhoI 583L Fwd CGCGGATCCCATATG-ATAGTTGACCAAAGCC 368 NdeI Rev CCCGCTCGAG-TTATTTTTCCGATTTTTCGG 369 XhoI 593 Fwd GCGGCCATATG-CTTGAACTGAACGGACT 370 NdeI Rev GCGGCCTCGAG-TCAGCGGAAGCGGACGATT 371 XhoI 650 (His-GST) Fwd CGCGGATCCCATATGTCCAAACTCAAAACCATCG 372 BamHI-NdeI (MC58) Rev CCCGCTCGAGGCTTCCAATCAGTTTGACC 373 XhoI 652 Fwd GCGGCCATATG-AGCGCAATCGTTGATATTTTC 374 NdeI Rev GCGGCCTCGAG-TTATTTGCCCAGTTGGTAGAATG 375 XhoI 664L Fwd GCGGCCATATG-GTGATACATCCGCACTACTTC 376 NdeI Rev GCGGCCTCGAG-TCAAAATCGAGTTTTACACCA 377 XhoI 726 Fwd GCGGCCATATG-ACCATCTATTTCAAAAACGG 378 NdeI Rev GCGGCCTCGAG-TCAGCCGATGTTTAGCGTCCATT 379 XhoI 741-His (MC58) Fwd CGCGGATCCCATATG-AGCAGCGGAGGGGGTG 380 NdeI Rev CCCGCTCGAG-TTGCTTGGCGGCAAGGC 381 XhoI ΔG741-His (MC58) Fwd CGCGGATCCCATATG-GTCGCCGCCGACATCG 382 NdeI Rev CCCGCTCGAG-TTGCTTGGCGGCAAGGC 383 XhoI 686-2-(His/GST) Fwd CGCGGATCCCATATG-GGCGGTTCGGAAGGCG 384 BamHI-NdeI (MC58) Rev CCCGCTCGAG-TTGAACACTGATGTCTTTTCCGA 385 XhoI 719-(His/GST) Fwd CGCGGATCCGCTAGC-AAACTGTCGTTGGTGTTAAC 386 BamHI-NheI (MC58) Rev CCCGCTCGAG-TTGACCCGCTCCACGG 387 XhoI 730-His (MC58) Fwd GCCGCCATATGGCGGACTTGGCGCAAGACCC 388 NdeI Rev GCCGCCTCGAGATCTCCTAAACCTGTTTTAACAATGCCG 389 XhoI 730A-His (MC58) Fwd GCCGCCATATGGCGGACTTGGCGCAAGACCC 390 NdeI Rev GCGGCCTCGAGCTCCATGCTGTTGCCCCAGC 391 XhoI 730B-His (MC58) Fwd GCCGCCATATGGCGGACTTGGCGCAAGACCC 392 NdeI Rev GCGGCCTCGAGAAAATCCCCGCTAACCGCAG 393 XhoI 741-His (MC58) Fwd CGCGGATCCCATATG-AGCAGCGGAGGGGGTG 394 NdeI Rev CCCGCTCGAG-TTGCTTGGCGGCAAGGC 395 XhoI ΔG741-His (MC58) Fwd CGCGGATCCCATATG-GTCGCCGCCGACATCG 396 NdeI Rev CCCGCTCGAG-TTGCTTGGCGGCAAGGC 397 XhoI 743 (His-GST) Fwd CGCGGATCCCATATGGACGGTGTTGTGCCTGTT 398 BamHI-NdeT Rev CCCGCTCGAGCTTACGGATCAAATTGACG 399 XhoI 757 (His-GST) Fwd CGCGGATCCCATATGGGCAGCCAATCTGAAGAA 400 BamHI-NdeI (MC58) Rev CCCGCTCGAGCTCAGCTTTTGCCGTCAA 401 XhoI 759-His/GST Fwd CGCGGATCCGCTAGC-TACTCATCCATTGTCCGC 402 BamHI-NheI (MC58) Rev CCCGCTCGAG-CCAGTTGTAGCCTATTTTG 403 XhoI 759L (MC58) Fwd CGCGGATCCGCTAGC-ATGCGCTTCACACACAC 404 NheI Rev CCCGCTCGAG-TTACCAGTTGTAGCCTATTT 405 XhoI 760-His Fwd GCCGCCATATGGCACAAACGGAAGGTTTGGAA 406 NdeI Rev GCCGCCTCGAGAAAACTGTAACGCAGGTTTGCCGTC 407 XhoI 769-His (MC58) Fwd GCGGCCATATGGAAGAAACACCGCGCGAACCG 408 NdeI Rev GCGGCCTCGAGGAACGTTTTATTAAACTCGAC 409 XhoI 907L Fwd GCGGCCATATG-AGAAAACCGACCGATACCCTA 410 NdeI Rev GCGGCCTCGAG-TCAACGCCACTGCCAGCGGTTG 411 XhoI 911L Fwd CGCGGATCCCATATG-AAGAAGAACATATTGGAATTTTGGGTCGGACTG 412 NdeI Rev CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG 413 XhoI 911LOmpA Fwd GGGAATTCCATATGAAAAAGACAGCTATCGCGATTGCA 414 NdeI-(NheI) GTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGC TAGC-GCTTTCCGCGTGGCCGGCGGTGC Rev CCCGCTCGAG-TTATTCGGCGGCTTTTTCGCATTGCCG 415 XhoI 911LPelB Fwd CATGCCATGG-CTTTCCGCGTGGCCGGCGGTGC 416 NcoI Rev CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG 417 XhoI 913-His/GST Fwd CGCGGATCCCATATG-TTTGCCGAAACCCGCC 418 BamHI-NdeI (MC58) Rev CCCGCTCGAG-AGGTTGTGTTCCAGGTTG 419 XhoI 913L (MC58) Fwd CGCGGATCCCATATG-AAAAAAACCGCCTATG 420 NdeI Rev CCCGCTCGAG-TTAAGGTTGTGTTCCAGG 421 XhoI 919L Fwd CGCGGATCCCATATG-AAAAAATACCTATTCCGC 422 NdeI Rev CCCGCTCGAG-TTACGGGCGGTATTCGG 423 XhoI 919 Fwd CGCGGATCCCATATG-CAAAGCAAGAGCATCCAAA 424 NdeI Rev CCCGCTCGAG-TTACGGGCGGTATTCGG 425 XhoI 919L Orf4 Fwd GGGAATTCCATATGAAAACCTTCTTCAAAACCCTTTCCG 426 NdeI-(NheI) CCGCCGCGCTAGCGCTCATCCTCGCCGCC- TGCCAAAGCAAGAGCATC Rev CCCGCTCGAG-TTACGGGCGGTATTCGGGCTTCATACCG 427 XhoI (919)-287fasion Fwd CGCGGATCCGTCGAC-TGTGGGGGCGGCGGTGGC 428 SalI Rev CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC 429 XhoI 920-1L Fwd GCGGCCATATG-AAGAAAACATTGACACTGC 430 NdeI Rev GCGGCCTCGAG-TTAATGGTGCGAATGACCGAT 431 XhoI 925-His/GST Fwd ggggacaagagtacaaaaaagcaggctTGCGGCAAGGATGCCGG 432 attB1 (MC58) ^(GATE) Rev ggggaccactagtacaagaaagctgggtCTAAAGCAACAATGCCGG 433 attB2 926L Fwd CGCGGATCCCATATG-AAACACACCGTATCC 434 NdeI Rev CCCGCTCGAG-TTATCTCGTGCGCGCC 435 XhoI 927.2-(His/GST) Fwd CGCGGATCCCATATG-AGCCCCGCGCCGATT 436 BamHI-NdeI (MC58) Rev CCCGCTCGAG-TTTTTGTGCGGTCAGGCG 437 XhoI 932-His/GST Fwd ggggacaagtttgtacaaaaaagcaggctTGTTCGTTTGGGGGATTTAA 438 attB1 (MC58) ^(GATE) ACCAAACCAAATC 935 (His-GST) For CGCGGATCCCATATGGCGGATGCGCCCGCG 439 BamHI-NdeI (MC58) Rev CCCGCTCGAGAAACCGCCAATCCGCC 440 XhoI 936-1L Rev ggggaccactagtacaagaaagagggtTCATTTTGTTTTTCCTTCTTCT 441 attB2 CGAGGCCATT Fwd CGCGGATCCCATATG-AAACCCAAACCGCAC 442 NdeI Rev CCCGCTCGAG-TCAGCGTTGGACGTAGT 443 Xhol 953L Fwd GGGAATTCCATATG-AAAAAAATCATCTTCGCCG 444 NdeI Rev CCCGCTCGAG-TTATTGTTTGGCTGCCTCGAT 445 XhoI 953-fa Fwd GGGAATTCCATATG-GCCACCTACAAAGTGGACG 446 NdeI Rev CGGGGATCC-TTGTTTGGCTGCCTCGATTTG 447 BamHI 954 (His-GST) Fwd CGCGGATCCCATATGCAAGAACAATCGCAGAAAG 448 BamHI-NdeI (MC58) Rev CCCGCTCGAGTTTTTTCGGCAAATTGGCTT 449 XhoI 958-His/GST Fwd ggggacaagtttgtacaaaaaagcaggctGCCGATGCCGTTGCGG 450 attB1 (MC58) ^(GATE) Rev ggggaccactttgtacaagaaagctgggcTCAGGGTCGTTGTTGCG 451 attB2 961L Fwd CGCGGATCCCATATG-AAACACTTTCCATCC 452 NdeI Rev CCCGCTCGAG-TTACCACTCGTAATTGAC 453 XhoI 961 Fwd CGGATCCCATATG-GCCACAAGCGACGAC 454 NdeI Rev CCCGCTCGAG-TTACCACTCGTAATTGAC 455 XhoI 961 c (His/GST) Fwd CGCGGATCCCATATG-GCCACAAACGACG 456 BamHI-NdeI Rev CCCGCTCGAG-ACCCACGTTGTAAGGTTG 457 XhoI 961 c-(His/GST) Fwd CGCGGATCCCATATG-GCCACAAGCGACGACGA 458 BamHI-NdeI (MC58) Rev CCCGCTCGAG-ACCCACGTTGTAAGGTTG 459 XhoI 961 c-L Fwd CGCGGATCCCATATG-ATGAAACACTTTCCATCC 460 Ndel Rev CCCGCTCGAG-TTAACCCACGTTGTAAGGT 461 XhoI 961 c-L (MC58) Fwd CGCGGATCCCATATG-ATGAAACACTTTCCATCC 462 NdeI Rev CCCGCTCGAG-TTAACCCACGTTGTAAGGT 463 XhoI 961 d (His/GST) Fwd CGCGGATCCCATATG-GCCACAAACGACG 464 BamHI-NdeI Rev CCCGCTCGAG-GTCTGACACTGTTTTATCC 465 XhoI 961 Δ1-L Fwd CGCGGATCCCATATG-ATGAAACACTTTCCATCC 466 NdeI Rev CCCGCTCGAG-TTATGCTTTGGCGGCAAAG 467 XhoI fu 961- . . . Fwd CGCGGATCCCATATG-GCCACAAACGACGAC 468 NdeI Rev CGCGGATCC-CCACTCGTAATTGACGCC 469 BamHI fu 961- . . . Fwd CGCGOATCCCATATG-GCCACAAOCGACGAC 470 NdeI (MC58) Rev CGCGGATCC-CCACTCGTAATTGACGCC 471 BamHI fu 961 c- . . . Fwd CGCGGATCCCATATG-GCCACAAACGACGAC 472 NdeI Rev CGCGGATCC-ACCCACGTTGTAAGGTTG 473 BamHI fu 961 c-L- . . . Fwd CGCGGATCCCATATG-ATGAAACACTTTCCATCC 474 NdeI Rev CGCGGATCC-ACCCACCTTTGTAAGGTTG 475 BamHI fu (961)-741 Fwd CGCGGATCC-GGAGGGGGTGGTGTCG 476 BamHI (MC58)-His Rev CCCGCTCGAG-TTGCTTGGCGGCAAGGC 477 XhoI fu (961)-983-His Fwd CGCGGATCC-GGCGGAGGCGGCACTT 478 BamHI Rev CCCGCTCGAG-GAACCGGTAGCCTACG 479 XhoI fu (961)-Orf46.1- Fwd CGCGGATCCGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC 480 BamHI His Rev CCCGCTCGAG-CGTATCATATTTCACGTGC 481 XhoI fu (961 c-L)-741 Fwd CGCGGATCC-GGAGGGGGTGGTGTCG 482 BamHI (MC58) Rev CCCGCTCGAG-TTATTGCTTGGCGGCAAG 483 XhoI fu (961e-L )-983 Fwd CGCGGATCC-GGCGGAGGCGGCACTT 484 BamHI Rev CCCGCTCGAG-TCAGAACCGGTAGCCTAC 485 XhoI fu (961c-L)- Fwd CGCGGATCCGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC 486 BamHI Orf46.1 Rev CCCGCTCGAG-TTACGTATCATATTTCACGTGC 487 XhoI 961-(His/GST) Fwd CGCGGATCCCATATG-GCCACAAGCGACGACG 488 BamHI-NdeI (MC58) Rev CCCGCTCGAG-CCACTCGTAATTGACGCC 489 XhoI 961 Δ1-His Fwd CGCGGATCCCATATG-GCCACAAACGACGAC 490 NdeI Rev CCCGCTCGAG-TGCTTTGGCGGCAAAGTT 491 XhoI 961a-(His/GST) Fwd CGCGGATCCCATATG-GCCACAAACGACGAC 492 BamHI-NdeI Rev CCCGCTCGAG-TTTAGCAATATTATCTTTGTTCGTAGC 493 XhoI 961b-(His/GST) Fwd CGCGGATCCCATATG-AAAGCAAACCGTGCCGA 494 BamHI-NdeI Rev CCCGCTCGAG-CCACTCGTAATTGACGCC 495 XhoI 961-His/GST ^(GATE) Fwd ggggacaagtttgtacaaaaaagcaggctGCAGCCACAAACGACGACG 496 attB1 ATGTTAAAAAAGC Rev ggggaccactttgtacaagaaagctgggtTTACCACTCGTAATTGACGC 497 attB2 CGACATGGTAGG 982 Fwd GCGGCCATATG-GCAGCAAAAGACGTACAGTT 498 NdeI Rev GCGGCCTCGAG-TTACATCATGCCGCCCATACCA 499 XhoI 983-His (2996) Fwd CGCGGATCCGCTAGC-TTAGGCGGCGGCGGAG 500 NheI Rev CCCGCTCGAG-GAACCGGTAGCCTACG 501 XhoI ΔG983-His (2996) Fwd CCCCTAGCTAGC-ACTTCTGCGCCCGACTT 502 NheI Rev CCCGCTCGAG-GAACCGGTAGCCTACG 503 XhoI 983-His Fwd CGCGGATCCGCTAGC-TTAGGCGGCGGCGGAG 504 NheI Rev CCCGCTCGAG-GAACCGGTAGCCTACG 505 XhoI ΔG983-His Fwd CGCGGATCCGCTAGC-ACTTCTGCGCCCGACTT 506 NheI Rev CCCGCTCGAG-GAACCGGTAGCCTACG 507 XhoI 983L Fwd CGCGGATCCGCTAGC-CGAACGACCCCAACCTTCCCTACAAAAACTTTCAA 508 NheI Rev CCCGCTCGAG-TCAGAACCGACGTGCCAAGCCGTTC 509 XhoI 987-His (MC58) Fwd GCCGCCATATGCCCCCACTGGAAGAACGGACG 510 NdeI Rev GCCGCCTCGAGTAATAAACCTTCTATGGGCAGCAG 511 XhoI 989-(His/GST) Fwd CGCGGATCCCATATG-TCCGTCCACGCATCCG 512 BamHI-NdeI (MC58) Rev CCCGCTCGAG-TTTGAATTTGTAGGTGTATTG 513 XhoI 989L (MC58) Fwd CGCGGATCCCATATG-ACCCCTTCCGCACT 514 NdeI Rev CCCGCTCGAG-TTATTTGAATTTGTAGGTGTAT 515 XhoI CrgA-His (MC58) Fwd CGCGGATCCCATATG-AAAACCAATTCAGAAGAA 513 NdeI Rev CCCGCTCGAG-TCCACAGAGATTGTTTCC 517 XhoI PilC1-ES (MC58) Fwd GATGCCCGAAGGGCGGG 518 Rev GCCCAAGCTT-TCAGAAGAAGACTTCACGC 519 PilC1-His (MC58) Fwd CGCGGATCCCATATG-CAAACCCATAAATACGCTATT 520 NdeI Rev GCCCAAGCTT-GAAGAAGACTTCACGCCAG 521 HindIII Δ1PilC1-His Fwd CGCGGATCCCATATG-GTCTTTTTCGACAATACCGA 522 NdeI (MC58) Rev GCCCAAGCTT- 523 HindIII Pi1C1L (MC58) Fwd CGCGGATCCCATATG-AATAAAACTTTAAAAAGGCGG 524 NdeI Rev GCCCAAGCTT-TCAGAAGAAGACTTCACGC 525 HindIII ΔGTbp2-His (MC58) Fwd CGCGAATCCCATATG-TTCGATCTTGATTCTGTCGA 526 NdeI Rev CCCGCTCGAG-TCGCACAGGCTGTTGGCG 527 XhoI Tbp2-His (MC58) Fwd CGCGAATCCCATATG-TTGGGCGGAGGCGGCAG 528 NdeI Rev CCCGCTCGAG-TCGCACAGGCTGTTGGCG 529 XhoI Tbp2-His (MC58) Fwd CGCGAATCCCATATG-TTGGGCGGAGGCGGCAG 530 NdeI Rev CCCGCTCGAG-TCGCACAGGCTGTTGGCG 531 XhoI NMB0109-(His/GST) Fwd CGCGGATCCCATATG-GCAAATTTGGAGGTGCGC 532 BamHI-NdeI (MCS8) Rev CCCGCTCGAG-TTCGGAGCGGTTGAAGC 533 XhoI NMB0109L (MC58) Fwd CGCGGATCCCATATG-CAACGTCGTATTATAACCC 534 NdeI Rev CCCGCTCGAG-TTATTCGGAGCGGTTGAAG 535 XhoI NMB0207-(His/GST) Fwd CGCGGATCCCATATG-GGCATCAAAGTCGCCATCAACGGCTAC 536 BamHI-NdeI (MC58) Rev CCCGCTCGAG-TTTGAGCGGGCGCACTTCAAGTCCG 537 XhoI NMB0462-(His/GST) Fwd CGCGGATCCCATATG-GGCGGCAGCGAAAAAAAC 538 BamHI-NdeI (MC58) Rev CCCGCTCGAG-GTTGGTGCCGACTTTGAT 539 XhoI NMB0623-(His/GST) Fwd CGCGGATCCCATATG-GGCGGCGGAAGCGATA 540 BamHI-NdeI (MC58) Rev CCCGCTCGAG-TTTGCCCGCTTTGAGCC 541 XhoI NMB0625 (His-GST) Fwd CGCGGATCCCATATGGGCAAATCCGAAAATACG 542 BamHI-NdeI (MC58) Rev CCCGCTCGAGCATCCCGTACTGTTTCG 543 XhoI NMB0634 (His/GST)  Fwd ggggacaagtttgtacaaaaaagcaggctCCGACATTACCGTGTACAAC 544 attB1 (MC58) GGCCAACAAAGAA Rev ggggaccactagtacaagaaagctgggtCTTATTTCATACCGGCTTGCT 545 attB2 CAAGCAGCCGG NMB0776-His/GST Fwd ggggacaagtttgtacaaaaaagcaggctGATACGGTGTTTTCCTGTAA 546 attB1 (MC58) ^(GATE) AACGGACAACAA Rev ggggaccactttgtacaagaaagctgggtCTAGGAAAAATCGTCATCGT 547 attB2 TGAAATTCGCC NMB1115-His/GST Fwd ggggacaagtttgtacaaaaaagcaggctATGCACCCCATCGAAACC 548 attB1 (MC58) ^(GATE) Rev ggggaccactttgtacaagaaagctgggtCTAGTCTTGCAGTGCCTC 549 attB2 NMB1343-(His/GST) Fwd CGCGGATCCCATATG-GGAAATTTCTTATATAGAGGCATTAG 550 BamHI-NdeI (MC58) Rev CCCGCTCGAG-GTTAATTTCTATCAACTCTTTAGCAATAAT 551 XhoI NMB1369 (His-GST Fwd CGCGGATCCCATATGGCCTGCCAAGACGACA 552 BamHI-NdeI (MC58) Rev CCCGCTCGAGCCGCCTCCTGCCGAAA 553 XhoI NMB1551 (His-GST) Fwd CGCGGATCCCATATGGCAGAGATCTGTTGATAA 554 BamHI-NdeI (MC58) Rev CCCGCTCGAGCGGTTTTCCGCCCAATG 555 XhoI NMB1899 (His-GST) Fwd CGCGGATCCCATATGCAGCCGGATACGGTC 556 BamHI-NdeI (MC58) Rev CCCGCTCGAGAATCACTTCCAACACAAAAT 557 XhoI NMB2050-(His/GST) Fwd CGCGGATCCCATATG-TGGTTGCTGATGAAGGGC 558 BamHI-NdeI (MC58) Rev CCCGCTCGAG-GACTGCTTCATCTTCTGC 559 XhoI NMB2050L Fwd CGCGGATCCCATATG-GAACTGATGACTGTITTGC 560 NdeI (MC58) Rev CCCGCTCGAG-TCAGACTGCTTCATCTTCT 561 XhoI NMB2159-(His/GST) Fwd CGCGGATCCCATATG-AGCATTAAAGTAGCGATTAACGGTTTCGGC 562 BamHI-NdeI (MC58) Rev CCCGCTCGAG-GATTTTCCTGCGAAGTATTCCAAAGTGCG 563 XhoI fu-ΔG287-His Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC 564 NheI Rev CGGGGATCC-ATCCTGCTCTTTTTTGCCGG 565 BamHI fu-(ΔG287)-919- Fwd CGCGGATCCGGTGGTGGTGGT-CAAAGCAAGAGCATCCAAACC 566 BamHI His Rev CCCAAGCTT-TTCGGGCGGTATTCGGGCTTC 567 HindIII fu-(ΔG287)-953- Fwd CGCGGATCCGGTGGTGGTGGT-GCCACCTACAAAGTGGAC 568 BamHI His Rev GCCCAAGCTT-TTGTTTGGCTGCCTCGAT 569 HindIII fu-(ΔG287)-961- Fwd CGCGGATCCGGTGGTGGTGGT-ACAAGCGACGACG 570 BamHI His Rev GCCCAAGCTT-CCACTCGTAATTGACGCC 571 HindIII fu-(ΔG287)- Fwd CGCGGATCCGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC 572 BamHI Orf46.1-His Rev CCCAAGCTT-CGTATCATATTTCACGTGC 573 HindIII fu-(ΔG287-919)- Fwd CCCAAGCTTGGTGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC 574 HindIII Orf46.1-His Rev CCCGCTCGAG-CGTATCATATTTCACGTGC 575 XhoI fu-(ΔG287-Orf46.1)- Fwd CCCAAGCTTGGTGGTGGTGGTGGT-CAAAGCAAGAGCATCCAAACC 576 HindII 919-His Rev CCCGCTCGAG-CGGGCGGTATTCGGGCTT 577 XhoI fu ΔG287 Fwd CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC 578 NheI (394.98)- . . . Rev CGGGGATCC-ATCCTGCTCTTTTTTGCCGG 579 BamHI fu Orf1-(Orf46.1)- Fwd CGCGGATCCGCTAGC-GGACACACTTATTTCGGCATC 580 NheI His Rev CGCGGATCC-CCAGCGGTAGCCTAATTTGAT 581 fu (Orf1)-Orf46.1- Fwd CGCGGATCCGGTGGTGGTGGT-TCAGATTTGGCAAACGATTC 582 BamHI His Rev CCCAAGCTT-CGTATCATATTTCACGTGC 583 HindIII fu (919)-Orf46.1- Fwd1 GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAG 584 SalI His Fwd2 GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC 585 Rev CCCGCTCGAG-CGTATCATATTTCACGTGC 586 XhoI Fu orf46- . . .  Fwd GGAATTCCATATGTCAGATTTGGCAAACGATTC 587 NdeI Rev CGCGGATCCGTATCATATTTCACGTGC 588 BamHI Fu (orf46)-287-His Fwd CGGGGATCCGGGGGCGGCGGTGGCG 589 BamHI Rev CCCAAGCTTATCCTGCTCTTTTTTGCCGGC 590 HindIII Fu (orf46)-919-His Fwd CGCGGATCCGGTGGTGGTGGTCAAAGCAAGAGCATCCAAACC 591 BamHI Rev CCCAAGCTTCGGGCGGTATTCGGGCTTC 592 HindIII Fu (orf46-919)- Fwd CCCCAAGCTTGGGGGCGGCGGTGGCG 593 HindIII 287-His Rev CCCGCTCGAGATCCTGCTCTTTTTTGCCGGC 594 XhoI Fu (orf46-287)- Fwd CCCAAGCTTGGTGGTGGTGGTGGTCAAAGCAAGAGCATCCAAACC 595 HindIII 919-His Rev CCCGCTCGAGCGGGCGGTATTCGGGCTT 596 XhoI (ΔG741)-961c-His Fwd1 GGAGGCACTGGATCCGCAGCCACAAACGACGACGA 597 XhoI Fwd2 GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG 598 Rev CCCGCTCGAG-ACCCAGCTTGTAAGGTTG 599 XhoI (ΔG741)-961-His Fwd1 GGAGGCACTGGATCCGCAGCCACAAACGACGACGA 600 XhoI Fwd2 GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG 601 Rev CCCGCTCGAG-CCACTCGTAATTGACGCC 602 XhoI (ΔG741)-983-His Fwd GCGGCCTCGAG-GGATCCGGCGGAGGCGGCACTTCTGCG 603 XhoI Rev CCCGCTCGAG-GAACCGGTAGCCTACG 604 XhoI (ΔG741)-orf46.1- Fwd1 GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC 605 SalI His Fwd2 GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA 606 Rev CCCGCTCGAG-CGTATCATATTTCACGTGC 607 XhoI (ΔG983)-741 Fwd GCGGCCTCGAG-GGATCCGGAGGGGGTGGTGTCGCC 608 XhoI (MC58)-His Rev CCCGCTCGAG-TTGCTTGGCGGCAAG 609 XhoI (ΔG983)-961c-His Fwd1 GGAGGCACTGGATCCGCAGCCACAAACGACGACGA 610 XhoI Fwd2 GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG 611 Rev CCCGCTCGAG-ACCCAGCTTGTAAGGTTG 612 XhoI (ΔG983)-961-His Fwd1 GGAGGCACTGGATCCGCAGCCACAAACGACGACGA 613 XhoI Fwd2 GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG 614 Rev CCCGCTCGAG-CCACTCGTAATTGACGCC 615 XhoI (ΔG983)-Orf46.1- Fwd1 GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC 616 SalI His Fwd2 GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA 617 Rev CCCGCTCGAG-CGTATCATATTTCACGTGC 618 XhoI * This primer was used as a Reverse primer for all the C terminal fusions of 287 to the His-tag ^(§) Forward primers used in combination with the 287-His Reverse primer. NB-All PCR reactions use strain 2996 unless otherwise specified (e.g. strain MC58)

In all constructs starting with an ATG not followed by a unique NheI site, the ATG codon is part of the NdeI site used for cloning. The constructs made using NheI as a cloning site at the 5′ end (e.g. all those containing 287 at the N-terminus) have two additional codons (GCT AGC) fused to the coding sequence of the antigen.

Preparation of Chromosomal DNA Templates

N. meningitidis strains 2996, MC58, 394.98, 1000 and BZ232 (and others) were grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% w/v sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml of lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one CHCl₃/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes of ethanol, and collected by centrifugation. The pellet was washed once with 70% (v/v) ethanol and redissolved in 4.0 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA concentration was measured by reading OD₂₆₀.

PCR Amplification

The standard PCR protocol was as follows: 200 ng of genomic DNA from 2996, MC581000, or BZ232 strains or 10 ng of plasmid DNA preparation of recombinant clones were used as template in the presence of 401M of each oligonucletide primer, 400-800 M dNTPs solution, 1×PCR buffer (including 1.5 mM MgCl₂), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AMPLITAQ® DNA Polymerase kit, Boerhingher Mannheim EXPAND™ Long Template kit).

After a preliminary 3 minute incubation of the whole mix at 95° C., each sample underwent a two-step amplification: the first 5 cycles were performed using the hybridisation temperature that excluded the restriction enzyme tail of the primer (T_(m1)). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (T_(m2)). Elongation times, performed at 68° C. or 72° C., varied according to the length of the Orf to be amplified. In the case of Orf1 the elongation time, starting from 3 minutes, was increased by 15 seconds each cycle. The cycles were completed with a 10 minute extension step at 72° C.

The amplified DNA was either loaded directly on a 1% agarose gel. The DNA fragment corresponding to the band of correct size was purified from the gel using the Qiagen Gel Extraction Kit, following the manufacturer's protocol.

Digestion of PCR Fragments and of the Cloning Vectors

The purified DNA corresponding to the amplified fragment was digested with the appropriate restriction enzymes for cloning into pET-21b+, pET22b+ or pET-24b+. Digested fragments were purified using the QIAquick PCR purification kit (following the manufacturer's instructions) and eluted with either H₂O or 10 mM Tris, pH 8.5. Plasmid vectors were digested with the appropriate restriction enzymes, loaded onto a 1.0% agarose gel and the band corresponding to the digested vector purified using the Qiagen QIAquick Gel Extraction Kit.

Cloning

The fragments corresponding to each gene, previously digested and purified, were ligated into pET21b+, pET22b+ or pET-24b+. A molar ratio of 3:1 fragment/vector was used with T4 DNA ligase in the ligation buffer supplied by the manufacturer.

Recombinant plasmid was transformed into competent E. coli DH5 or HB101 by incubating the ligase reaction solution and bacteria for 40 minutes on ice, then at 37° C. for 3 minutes. This was followed by the addition of 800 μl LB broth and incubation at 37° C. for 20 minutes. The cells were centrifuged at maximum speed in an Eppendorf microfuge, resuspended in approximately 200 μl of the supernatant and plated onto LB ampicillin (100 mg/ml) agar.

Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37° C. in 4.0 ml of LB broth+100 μg/ml ampicillin. Cells were pelleted and plasmid DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions. Approximately 1 μg of each individual miniprep was digested with the appropriate restriction enzymes and the digest loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 kb DNA Ladder, GIBCO®). Positive clones were selected on the basis of the size of insert.

Expression

After cloning each gene into the expression vector, recombinant plasmids were transformed into E. coli strains suitable for expression of the recombinant protein. 1 μl of each construct was used to transform E. coli BL21-DE3 as described above. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, to give an OD₆₀₀ between 0.1 and 0.2. The flasks were incubated at 30° C. or at 37° C. in a gyratory water bath shaker until OD₆₀₀ indicated exponential growth suitable for induction of expression (0.4-0.8 OD). Protein expression was induced by addition of 1.0 mM IPTG. After 3 hours incubation at 30° C. or 37° C. the OD₆₀₀ was measured and expression examined. 1.0 ml of each sample was centrifuged in a microfuge, the pellet resuspended in PBS and analysed by SDS-PAGE and Coomassie Blue staining.

Gateway Cloning and Expression

Sequences labelled GATE were cloned and expressed using the GATEWAY Cloning Technology (GIBCO®-BRL). Recombinational cloning (RC) is based on the recombination reactions that mediate the integration and excision of phage into and from the E. coli genome, respectively. The integration involves recombination of the attP site of the phage DNA within the attB site located in the bacterial genome (BP reaction) and generates an integrated phage genome flanked by attL and attR sites. The excision recombines attL and attR sites back to attP and attB sites (LR reaction). The integration reaction requires two enzymes [the phage protein Integrase (Int) and the bacterial protein integration host factor (IHF)] (BP clonase). The excision reaction requires Int, IHF, and an additional phage enzyme, Excisionase (Xis) (LR clonase). Artificial derivatives of the 25-bp bacterial attB recombination site, referred to as B1 and B2, were added to the 5′ end of the primers used in PCR reactions to amplify Neisserial ORFs. The resulting products were BP cloned into a “Donor vector” containing complementary derivatives of the phage attP recombination site (P1 and P2) using BP clonase. The resulting “Entry clones” contain ORFs flanked by derivatives of the attL site (L1 and L2) and were subcloned into expression “destination vectors” which contain derivatives of the attL-compatible attR sites (R1 and R2) using LR clonase. This resulted in “expression clones” in which ORFs are flanked by B1 and B2 and fused in frame to the GST or His N terminal tags.

The E. coli strain used for GATEWAY expression is BL21-SI. Cells of this strain are induced for expression of the T7 RNA polymerase by growth in medium containing salt (0.3 M NaCl).

Note that this system gives N-terminus His tags.

Preparation of Membrane Proteins.

Fractions composed principally of either inner, outer or total membrane were isolated in order to obtain recombinant proteins expressed with membrane-localisation leader sequences. The method for preparation of membrane fractions, enriched for recombinant proteins, was adapted from Filip et. al. [J. Bact. (1973) 115:717-722] and Davies et. al. [J. Immunol. Meth. (1990) 143:215-225]. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at either 30° C. or 37° C. until the OD₅₅₀ reached 0.6-0.8. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. and resuspended in 20 ml of 20 mM Tris-HCl (pH 7.5) and complete protease inhibitors (Boehringer-Mannheim). All subsequent procedures were performed at 4° C. or on ice.

Cells were disrupted by sonication using a Branson SONIFIER® 450 (ultrasonic cell disruption/homogenizer) and centrifuged at 5000 g for 20 min to sediment unbroken cells and inclusion bodies. The supernatant, containing membranes and cellular debris, was centrifuged at 50000 g (Beckman Ti50, 29000 rpm) for 75 min, washed with 20 mM Bis-tris propane (pH 6.5), 1.0 M NaCl, 10% (v/v) glycerol and sedimented again at 50000 g for 75 minutes. The pellet was resuspended in 20 mM Tris-HCl (pH 7.5), 2.0% (v/v) Sarkosyl, complete protease inhibitor (1.0 mM EDTA, final concentration) and incubated for 20 minutes to dissolve inner membrane. Cellular debris was pelleted by centrifugation at 5000 g for 10 min and the supernatant centrifuged at 75000 g for 75 minutes (Beckman Ti50, 33000 rpm). Proteins 008L and 519L were found in the supernatant suggesting inner membrane localisation. For these proteins both inner and total membrane fractions (washed with NaCl as above) were used to immunise mice. Outer membrane vesicles obtained from the 75000 g pellet were washed with 20 mM Tris-HCl (pH 7.5) and centrifuged at 75000 g for 75 minutes or overnight. The OMV was finally resuspended in 500 μl of 20 mM Tris-HCl (pH 7.5), 10% v/v glycerol. Orf1L and Orf40L were both localised and enriched in the outer membrane fraction which was used to immunise mice. Protein concentration was estimated by standard Bradford Assay (Bio-Rad), while protein concentration of inner membrane fraction was determined with the DC protein assay (Bio-Rad). Various fractions from the isolation procedure were assayed by SDS-PAGE.

Purification of his-Tagged Proteins

Various forms of 287 were cloned from strains 2996 and MC58. They were constructed with a C-terminus His-tagged fusion and included a mature form (aa 18-427), constructs with deletions (Δ1, Δ2, Δ3 and Δ4) and clones composed of either B or C domains. For each clone purified as a His-fusion, a single colony was streaked and grown overnight at 37° C. on a LB/Amp (100 μg/ml) agar plate. An isolated colony from this plate was inoculated into 20 ml of LB/Amp (100 μg/ml) liquid medium and grown overnight at 37° C. with shaking. The overnight culture was diluted 1:30 into 1.0 L LB/Amp (100 μg/ml) liquid medium and allowed to grow at the optimal temperature (30 or 37° C.) until the OD₅₅₀ reached 0.6-0.8. Expression of recombinant protein was induced by addition of IPTG (final concentration 1.0 mM) and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 8000 g for 15 min at 4° C. The bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8.0) for soluble proteins or (ii) buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8 and, optionally, 8M urea) for insoluble proteins. Proteins purified in a soluble form included 287-His, Δ1, Δ2, Δ3 and Δ4287-His, Δ4287MC58-His, 287c-His and 287cMC58-His. Protein 287bMC58-His was insoluble and purified accordingly. Cells were disrupted by sonication on ice four times for 30 sec at 40 W using a Branson SONIFIER® 450 (ultrasonic cell disruption/ homogenizer) and centrifuged at 13000×g for 30 min at 4° C. For insoluble proteins, pellets were resuspended in 2.0 ml buffer C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5 and treated with 10 passes of a Dounce homogenizer. The homogenate was centrifuged at 13000 g for 30 min and the supernatant retained. Supernatants for both soluble and insoluble preparations were mixed with 150 μl Ni²⁺-resin (previously equilibrated with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 min. The resin was Chelating SEPHAROSE™ Fast Flow (IMAC medium, Pharmacia), prepared according to the manufacturer's protocol. The batch-wise preparation was centrifuged at 700 g for 5 min at 4° C. and the supernatant discarded. The resin was washed twice (batch-wise) with 10 ml buffer A or B for 10 min, resuspended in 1.0 ml buffer A or B and loaded onto a disposable column. The resin continued to be washed with either (i) buffer A at 4° C. or (ii) buffer B at room temperature, until the OD₂₈₀ of the flow-through reached 0.02-0.01. The resin was further washed with either (i) cold buffer C (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8.0) or (ii) buffer D (10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3 and, optionally, 8M urea) until OD₂₈₀ of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8.0) or (ii) elution buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5 and, optionally, 8M urea) and fractions collected until the OD₂₈₀ indicated all the recombinant protein was obtained. 20 μl aliquots of each elution fraction were analysed by SDS-PAGE. Protein concentrations were estimated using the Bradford assay.

Renaturation of Denatured his-Fusion Proteins.

Denaturation was required to solubilize 287bMC8, so a renaturation step was employed prior to immunisation. Glycerol was added to the denatured fractions obtained above to give a final concentration of 10% v/v. The proteins were diluted to 200 μg/ml using dialysis buffer I (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, 2.0M urea, pH 8.8) and dialysed against the same buffer for 12-14 hours at 4° C. Further dialysis was performed with buffer II (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was estimated using the formula: Protein(mg/ml)=(1.55×OD ₂₈₀)−(0.76×OD ₂₆₀) Amino Acid Sequence Analysis.

Automated sequence analysis of the NH₂-terminus of proteins was performed on a Beckman sequencer (LF 3000) equipped with an on-line phenylthiohydantoin-amino acid analyser (System Gold) according to the manufacturer's recommendations.

Immunization

Balb/C mice were immunized with antigens on days 0, 21 and 35 and sera analyzed at day 49.

Sera Analysis—ELISA

The acapsulated MenB M7 and the capsulated strains were plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO₂. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.4-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and bacteria were washed twice with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 1 hour at 37° C. and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% TWEEN®-20 (Polysorbate 20, Sigma Aldrich) in PBS). 200 μl of saturation buffer (2.7% polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer: 1% BSA, 0.1% TWEEN®-20 (Polysorbate 20, Sigma Aldrich), 0.1% NaN₃ in PBS) were added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 μl of H₂SO₂) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl 12.5% H₂SO₄ was added to each well and OD₄₉₀ was followed. The ELISA titers were calculated arbitrarily as the dilution of sera which gave an OD₄₉₀ value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD₄₉₀ of 0.4 was higher than 1:400.

Sera Analysis—FACS Scan Bacteria Binding Assay

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO₂. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA in PBS, 0.4% NaN₃) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD₆₂₀ of 0.05. 100 μl bacterial cells were added to each well of a COSTAR®96 well plate. 100 μl of diluted (1:100, 1:200, 1:400) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytrin conjugated F(ab)₂ goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan (Laser Power 15 mW) setting were: FL2 on; FSC-H threshold: 92; FSC PMT Voltage: E 01; SSC PMT: 474; Amp. Gains 6.1; FL-2 PMT: 586; compensation values: 0.

Sera Analysis—Bactericidal Assay

N. meningitidis strain 2996 was grown overnight at 37° C. on chocolate agar plates (starting from a frozen stock) with 5% CO₂. Colonies were collected and used to inoculate 7 ml Mueller-Hinton broth, containing 0.25% glucose to reach an OD₆₂₀ of 0.05-0.08. The culture was incubated for approximately 1.5 hours at 37 degrees with shacking until the OD₆₂₀ reached the value of 0.23-0.24. Bacteria were diluted in 50 mM Phosphate buffer pH 7.2 containing 10 mM MgCl₂, 10 mM CaCl₂ and 0.5% (w/v) BSA (assay buffer) at the working dilution of 10⁵ CFU/ml. The total volume of the final reaction mixture was 50 μl with 25 μl of serial two fold dilution of test serum, 12.5 μl of bacteria at the working dilution, 12.5 μl of baby rabbit complement (final concentration 25%).

Controls included bacteria incubated with complement serum, immune sera incubated with bacteria and with complement inactivated by heating at 56° C. for 30′. Immediately after the addition of the baby rabbit complement, 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 0). The 96-wells plate was incubated for 1 hour at 37° C. with rotation. 7 μl of each sample were plated on Mueller-Hinton agar plates as spots, whereas 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 1). Agar plates were incubated for 18 hours at 37 degrees and the colonies corresponding to time 0 and time 1 were counted.

Sera Analysis—Western Blots

Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded onto a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., using transfer buffer (0.3% Tris base, 1.44% glycine, 20% (v/v) methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

The OMVs were prepared as follows: N. meningitidis strain 2996 was grown overnight at 37 degrees with 5% CO₂ on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20 mM Tris-HCl pH 7.5, 2 mM EDTA. Heat inactivation was performed at 56° C. for 45 minutes and the bacteria disrupted by sonication for 5 minutes on ice (50% duty cycle, 50% output, Branson SONIFIER®3 mm microtip). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes, the supernatant containing the total cell envelope fraction recovered and further centrifuged overnight at 50000 g at the temperature of 4° C. The pellet containing the membranes was resuspended in 2% sarkosyl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA and incubated at room temperature for 20 minutes to solubilise the inner membranes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, the supernatant was further centrifuged at 50000 g for 3 hours. The pellet, containing the outer membranes was washed in PBS and resuspended in the same buffer. Protein concentration was measured by the D.C. Bio-Rad Protein assay (Modified Lowry method), using BSA as a standard.

Total cell extracts were prepared as follows: N. meningitidis strain 2996 was grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.

961 Domain Studies

Cellular Fractions Preparation Total lysate, periplasm, supernatant and OMV of E. coli clones expressing different domains of 961 were prepared using bacteria from over-night cultures or after 3 hours induction with IPTG. Briefly, the periplasm were obtained suspending bacteria in saccarose 25% and Tris 50 mM (pH 8) with polimixine 100 μg/ml. After 1 hr at room temperature bacteria were centrifuged at 13000 rpm for 15 min and the supernatant were collected. The culture supernatant were filtered with 0.2 μm and precipitated with TCA 50% in ice for two hours. After centrifugation (30 min at 13000 rp) pellets were rinsed twice with ethanol 70% and suspended in PBS. The OMV preparation was performed as previously described. Each cellular fraction were analyzed in SDS-PAGE or in Western Blot using the polyclonal anti-serum raised against GST-961.

Adhesion Assay Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, human conjunctiva) were maintained in DMEM (GIBCO®) supplemented with 10% heat-inactivated FCS, 15 mM L-glutamine and antibiotics.

For the adherence assay, sub-confluent culture of Chang epithelial cells were rinsed with PBS and treated with trypsin-EDTA (GIBCO®), to release them from the plastic support. The cells were then suspended in PBS, counted and dilute in PBS to 5×10⁵ cells/ml.

Bacteria from over-night cultures or after induction with IPTG, were pelleted and washed twice with PBS by centrifuging at 13000 for 5 min. Approximately 2−3×10⁸ (cfu) were incubated with 0.5 mg/ml FITC (Sigma) in 1 ml buffer containing 50 mM NaHCO₃ and 100 mM NaCl pH 8, for 30 min at room temperature in the dark. FITC-labeled bacteria were wash 2-3 times and suspended in PBS at 1−1.5×10⁹/ml. 200 μl of this suspension (2-3×10⁸) were incubated with 200 μl (1×10⁵) epithelial cells for 30 min a 37° C. Cells were than centrifuged at 2000 rpm for 5 min to remove non-adherent bacteria, suspended in 200 μl of PBS, transferred to FACScan tubes and read 

We claim:
 1. A method of expressing an E. coli lipidated protein comprising a) providing an E. coli host cell containing a nucleic acid encoding a heterologous leader peptide operably linked to a coding sequence for the protein, wherein the protein comprises i) a sequence having greater than 80% sequence identity to SEQ ID NO: 634; or ii) a fragment of at least 14 consecutive amino acids from SEQ ID NO: 634, wherein the E. coli host cell is capable of lipidating the protein and the heterologous leader peptide directs lipidation in the E. coli host cell; b) expressing of the protein under conditions where the protein is lipidated at an N-terminal cysteine.
 2. The method of claim 1, wherein the protein comprises (i).
 3. The method of claim 2, wherein the sequence has greater than 90% sequence identity to SEQ ID NO:
 634. 4. The method of claim 2 further comprising c) isolating the E. coli lipidated protein from the E. coli host cell.
 5. The method of claim 4 further comprising d) formulating an immunogenic composition with the E. coli lipidated protein isolated from the E. coli host cell.
 6. The method of claim 5, wherein the immunogenic composition further comprises an alum adjuvant.
 7. The method of claim 1, wherein the protein comprises (ii).
 8. The method of claim 7, wherein the fragment includes 20 or more contiguous amino acids from SEQ ID NO:
 634. 9. The method of claim 8, wherein the fragment includes 50 or more contiguous amino acids from SEQ ID NO:
 634. 10. The method of claim 8 further comprising c) isolating the E. coli lipidated protein from the E. coli host cell.
 11. The method of claim 10 further comprising d) formulating an immunogenic composition with the E. coli lipidated protein isolated from the E. coli host cell.
 12. The method of claim 11, wherein the immunogenic composition further comprises an alum adjuvant. 